Regulated gene expression in plants

ABSTRACT

A method is provided of regulating transcription in a plant cell from a DNA sequence comprising a target DNA operably linked to a coding sequence, which method comprises introducing an engineered zinc finger polypeptide in said plant cell which polypeptide binds to the target DNA and modulates transcription of the coding sequence.

REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE

[0001] This application is a continuation-in-part of PCT application no.PCT/GB00/02071 entitled “GENE SWITCHES” filed May 30, 2000 designatingthe US and claiming priority from GB applications 9912635.1 filed May18, 1999 and 001578.4 filed Jan. 24, 2000. Further mentioned andincorporated by reference herein are PCT/GB99/03730, filed Nov. 9, 1999,published as WO00/27878A1 on May 18, 2000 entitled “Screening System ForZinc Finger Polypeptides For A Desired Binding Ability” and claimingpriority from GB application 9824544.2, filed Nov. 9, 1998, anddesignating the US; PCT/GB99/03730 which is a continuation-in-part ofU.S. patent application Ser. No. 09/139,672, filed Aug. 25, 1998 (nowU.S. Pat. No. 6,013,453), which is a continuation of U.S. patentapplication Ser. No. 08/793,408 (now U.S. Pat. No. 6,007,988), filed asPCT application no. PCT/GB95/01949 on Aug. 17, 1995, designating theU.S. and, published as WO96/06166 on Feb. 29, 2996 entitled“Improvements in or Relating to Binding Proteins for Recognition ofDNA”; PCT/GB95/01949 claims the benefit of priority from GB application9514698.1, filed Jul. 19, 1995, GB application 9422534.9, filed Nov. 8,1994 and GB application no. 9416880.4, filed Aug. 20, 1994. Mention isalso made of: U.S. Ser. No. 08/422,107; WO96/32475; WO99/47656A2,published Sep. 23, 1999 entitled “Nucleic Acid Binding Proteins”;WO98/53060A1, published Nov. 26, 1998 entitled “Nucleic Acid BindingProteins”; WO98/53059A1 published Nov. 26, 1998 entitled “Nucleic AcidBinding Proteins”; WO98/53058A1 published Nov. 26, 1998 entitled“Nucleic Acid Binding Proteins”); WO98/53057A1 published Nov. 26, 1998(“Nucleic Acid Binding Polypeptide Library”; U.S. Pat. Nos. 6,013,453and 6,007,988; Fichn et al. (2000) Nature Biotechnol. 18:1157-1161;Richter et al. (2000) Nature Biotechnol. 18:1167-1171; and, generally,Nature Biotechnol. Vol. 18(11) together with all documents cited orreferenced therein. Each of the foregoing applications and patents, andeach document cited or referenced in each of the foregoing applicationsand patents, including during the prosecution of each of the foregoingapplications and patents (“application cited documents”) and anymanufacturer 's instructions or catalogues for any products cited ormentioned in each of the foregoing applications and patents and in anyof the application cited documents, are hereby incorporated herein byreference. Furthermore, all documents cited in this text, and alldocuments cited or referenced in documents cited in this text, and anymanufacturer's instructions or catalogues for any products cited ormentioned in this text, are hereby incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] Not applicable.

TECHNICAL FIELD

[0003] This invention relates to the regulation of gene expression inplants using engineered zinc fingers that bind to sequences within generegulatory sequences. Moreover, this invention also relates totransgenic plants that comprise engineered zinc finger-containingpeptides.

BACKGROUND OF THE INVENTION

[0004] The application of biotechnology to plants has yielded manyagricultural gains. For example, biotechnology has been used to improvevarious properties of plants such as resistance to pests and diseases,resistance to herbicides, and the improvement of various seed and fruittraits. Many further applications of plant biotechnology are anticipatedand these include the modification of specific traits that may be ofagronomic interest or of interest in the processing and use ofplant-derived products. In many instances, this could be undertaken bythe manipulation of endogenous genes which encode these traits, however,the sophisticated means to achieve up- and down-regulation of suchendogenous genes is, in many cases, not yet available. In addition,plants also hold great promise as biological “factories” for a varietyof chemical products including enzymes and compounds for industrial andpharmaceutical use. However, it is expected that the continuousproduction of high concentrations of gene products and compounds forsuch use may have deleterious consequences for the host plant andconsequently, more sophisticated mechanisms for expressing such genesare required.

[0005] Accordingly, gene switches are currently of interest for thecontrol of timing and/or dosage of gene expression in plants. Inparticular, the development of gene switches that can be directedtowards any gene in a plant chromosome is highly desirable..

SUMMARY OF THE INVENTION

[0006] The present invention seeks to overcome these difficulties byproviding non-naturally occurring engineered zinc finger proteins toconfer specificity on gene regulation for both endogenous genes andtransgenes of interest. More specifically, the present invention can beused to regulate any gene in a plant.

[0007] Accordingly the present invention provides a method of regulatingtranscription in a plant cell which method comprises introducing anengineered zinc finger polypeptide into said plant cell whichpolypeptide binds to a target DNA and modulates transcription of acoding sequence which is operably linked to said target DNA.

[0008] Previously, it has been reported that engineered zinc fingers canbe used to regulate genes in mammalian cells (see for example Choo etal., Nature 372: 642-645 (1994); Pomerantz et al, Science 267: 93-96(1995); Liu et al. PNAS 94: 5525-5530 (1997); Beerli et al. PNAS 95:14628-14633 (1998)). However, only in the case of Choo et al. was theregulated gene a gene integrated in a chromosome of the host mammaliancell. It is well recognized that the biology of mammalian and plantcells is very different and that each has evolved be very different atthe structural, physiological, biochemical and molecular biologicallevel. In the present invention, the inventors have shown for the firsttime that it is possible to regulate a gene in a plant using anengineered zinc finger. More specifically, the inventors have shownthat, using an engineered zinc finger, a gene integrated in a plantchromosome can be regulated in a via binding of the engineered zincfinger to a target DNA sequence adjacent to the target gene.

[0009] The zinc fingers of the present invention can be used toup-regulate or down-regulate any gene in a plant. By designing a zincfinger with a transactivating domain the induction of an endogenous genecan be accomplished specifically and bypass any endogenous regulation ofthe targeted gene. Previously, the only available method was tointroduce a transgene in another location of the genome under theregulation of a separate promoter. The zinc fingers of the presentinvention can also be used to down-regulate any gene in any plant, whichhas previously only been possible using techniques such as antisense,ribozymes and co-suppression, all of which are somewhat unpredictable.The zinc finger approach to down-regulation is highly potent and allowsthe targeting of specific member of a gene family without affecting theother members.

[0010] The term “engineered” means that the zinc finger has beengenerated or modified in vitro. It has therefore typically been producedby deliberate mutagenesis, for example the substitution of one or moreamino acids, either as part of a random mutagenesis procedure orsite-directed mutagenesis, or by selection from a library or librariesof mutated zinc fingers. Engineered zinc fingers for use in theinvention can also be produced de novo using rational design strategies.

[0011] The term “introduced into” means that a procedure is performed ona plant, a plant part, or a plant cell such that the zinc fingerpolypeptide is then present in the cell or cells. Examples of suitableprocedures include the microinjection, bombardment, agrobacteriumtransformation, electroporation, transfection or other transformation ordelivery techniques of cells with a nucleic acid construct that iscapable of directing expression of the zinc finger polypeptide in thecell, or the zinc finger protein itself.

[0012] The term “target DNA sequence” means any nucleic acid sequence towhich a zinc finger is capable of binding. It is usually but notnecessarily a DNA sequence within a plant chromosome, to which anengineered zinc finger is capable of binding. A target DNA sequence willgenerally be associated with a target gene (see below) and the bindingof the engineered zinc finger to the gene will generally allow the up-or down-regulation of the associated gene. In one embodiment, the targetDNA sequence is part of an endogenous genomic sequence. In anotherembodiment, the target DNA sequence and coding sequence have beenintroduced into the cell or are heterologous to the cell. In many cases,a target DNA sequence will form part of a promoter or othertranscription regulatory region such as an enhancer. In a most preferredembodiment, the target DNA is a known sequence of a promoter from aplant gene of interest.

[0013] The term “target gene” means a gene in a plant or plant cell theexpression of which one may wish to affect using the methods describedin the present invention. A target gene may be an endogenous gene (i.e.one which is normally found in genome of the plant or plant cell) or aheterologous gene (i.e. one that does not normally exist in the genomeof the plant or cell).

[0014] The term “heterologous to the cell” means that the sequence doesnot naturally exist in the genome of the cell but has been introducedinto the cell. A heterologous sequence can include a modified sequenceintroduced at any chromosomal site, or which is not integrated into achromosome, or which is introduced by homologous recombination such thatit is present in the genome in the same position as the native allele.

[0015] In a highly preferred embodiment, the zinc finger polypeptide isfused to a biological effector domain. The term “biological effectordomain” means any polypeptide that has a biological function andincludes enzymes and transcriptional regulatory domains or proteins, andadditional sequence such as nuclear localization sequences.

[0016] Preferably the zinc finger polypeptide is fused to atranscriptional activator domain or a transcriptional repressor domain.

[0017] In a further embodiment of the method of the invention the plantcell is part of a plant or can be regenerated into a plant and thetarget sequence is part of a regulatory sequence to which the nucleotidesequence of interest is operably linked.

[0018] The present invention further provides a plant cell comprising apolynucleotide encoding an engineered zinc finger polypeptide and atarget sequence to which the zinc finger polypeptide binds.

[0019] The present invention also provides a transgenic plant comprisinga polynucleotide encoding an engineered zinc finger polypeptide and atarget sequence to which the zinc finger polypeptide binds.

[0020] The present invention further provides a transgenic plantcomprising a polynucleotide encoding an engineered zinc fingerpolypeptide and a target sequence which is within a plant chromosome.

[0021] The present invention further provides a transgenic plantcomprising a polynucleotide encoding an engineered zinc fingerpolypeptide and a target sequence which is within the sequence of a genewhich is endogenous to the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a general depiction of plasmids described herein andtheir use in transforming plants with Agrobacterium.

[0023]FIG. 2 is a more specific depiction of production of positivetransgenic lines.

[0024]FIG. 3, pBA002 and pER8.

[0025]FIG. 4 depicts the component parts and final constructs ofreporter constructs, and pZVE1.

[0026]FIG. 5 shows transient expression of TFIIIAZIFVP16/VP64 andactivation of luciferase reporter construct in onion peels bybombardment-mediated transformation. In FIG. 5, a. isTFIIIAZIFVP16+4XBS-Luciferase, b. is TFIIIAZIFVP16+1XBS-Luciferase, c.is TFIIIAZIFVP64+4XBS-Luciferase, d. is TFIIIAZIFVP16+4XBS-Luciferase,e. is TFIIIAZIFVP64+4XBS-Luciferase, f. is 4XBS-Luciferase, g. is1XBS-Luciferase, and h. is KIN2-Luciferase.

[0027]FIG. 6 shows 17-β-Estradiol (estrogen) regulated expression ofluciferase. In FIG. 6, a. is pER8-TFIIIAZIFVP64+pIXBSluciferase (withestrogen), b. is pIXBSluciferase (with estrogen), c. ispER8-TFIIIAZIFVP64+pIXBSluciferase (without estrogen) and d. ispIXBSluciferase (without estrogen).

[0028]FIG. 7FIG. 6 shows 17-β-Estradiol regulated expression of GFP. InFIG. 7, a. is pER8-TFIIIAZIFVP64 +p4XBSGFP and b. ispER8-TFIIIAZIFVP16+p4XBSGFP

[0029]FIG. 8 depicts luciferase expression by induction ofpER8-TFIIIAZIFVP16 in T1 transgenic plant leaves containing thepBA4XBSLUC and pER8-TFIIIAZIFVP16+p4XBSLUC.

[0030]FIG. 9 depicts expression of 1XBSGFP and 1XBSMRFP in onion peels.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art (e.g., in cell culture, molecular genetics, nucleicacid chemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al. Molecular Cloning:A Laboratory Manual, 2d ed.(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. andAusubel et al. Short Protocols in Molecular Biology (1999) 4^(th) Ed,John Wiley & Sons, Inc.), chemical methods, pharmaceutical formulationsand delivery and treatment of patients.

[0032] A. Zinc Fingers

[0033] A zinc finger chimera is a transcription factor that comprises aDNA binding domain (comprising a number of zinc fingers), designed tobind specifically to any DNA sequence and one or more further domains.Usually, a nuclear localization domain is attached to the zinc fingerdomain to direct the chimera to the nucleus. Generally, the chimera alsoincludes an effector domain that can be a transactivation or repressiondomain to regulate the expression of the gene in question. Choo and Klug(1995) Curr. Opin. Biotech. 6:431-436; Choo and Klug (1997); Rebar andPabo (1994) Science 263:671-673; and Jamieson et al. (1994) Biochem.33:5689-5695. The zinc finger chimera may also preferably include otherdomains which may be advantageous within the context of the presentinvention. For example, DNA modifying domains (such as endonucleases andmethylases) can be added to the zinc finger domain, conferring to thezinc finger chimera the ability to regulate expression of the gene ofinterest or modify any DNA specifically. Wu et al. (1995) Proc. Natl.Acad. Sci. USA 92:344-348; Nahon and Raveh (1998); Smith et al. (1999;and Carroll et al. (1999). Zinc finger proteins of the Cys2-His2 classare preferred within the context of the present invention.

[0034] Zinc fingers are small protein domains that are able to recognizeand bind a nucleic acid triplet, or an overlapping quadruplet, in anucleic acid binding sequence. Preferably, there are 2 or more zincfingers, for example 2, 3, 4, 5 or 6 zinc fingers, in each bindingprotein. Advantageously, there are 3 or more zinc fingers in each zincfinger binding protein.

[0035] All of the DNA binding residue positions of zinc fingers, asreferred to herein, are numbered from the first residue in the α-helixof the finger, ranging from +1 to +9. “−1” refers to the residue in theframework structure immediately preceding the α-helix in a Cys2-His2zinc finger polypeptide. Residues referred to as “++” are residuespresent in an adjacent (C-terminal) finger. Where there is no C-terminaladjacent finger, “++” interactions do not operate.

[0036] Zinc finger polypeptides according to the present invention areengineered. That is, essentially “man-made”. Typically, zinc fingersaccording to the invention are produced by mutagenesis techniques ordesigned using rational design techniques. Zinc fingers can also beselected from randomized libraries using screening procedures, such asthose described below.

[0037] The present invention is in one aspect concerned with theproduction of what are essentially engineered DNA binding proteins. Inthese proteins, artificial analogues of amino acids may be used, toimpart the proteins with desired properties or for other reasons. Thus,the term “amino acid”, particularly in the context where “any aminoacid” is referred to, means any sort of natural or artificial amino acidor amino acid analogue that may be employed in protein constructionaccording to methods known in the art. Moreover, any specific amino acidreferred to herein may be replaced by a functional analogue thereof,particularly an artificial functional analogue. The nomenclature usedherein therefore specifically comprises within its scope functionalanalogues or mimetics of the defined amino acids.

[0038] The α-helix of a zinc finger binding protein aligns antiparallelto the nucleic acid strand, such that the primary nucleic acid sequenceis arranged 3′ to 5′ in order to correspond with the N terminal toC-terminal sequence of the zinc finger. Since nucleic acid sequences areconventionally written 5′ to 3′, and amino acid sequences N-terminus toC-terminus, the result is that when a nucleic acid sequence and a zincfinger protein are aligned according to convention, the primaryinteraction of the zinc finger is with the—strand of the nucleic acid,since it is this strand which is aligned 3′ to 5′. These conventions arefollowed in the nomenclature used herein. It should be noted, however,that in nature certain fingers, such as finger 4 of the protein GLI,bind to the + strand of nucleic acid: see Suzuki el al., (1994) NAR22:3397-3405 and Pavletich and Pabo, (1993) Science 261:1701-1707. Theincorporation of such fingers into DNA binding molecules according tothe invention is envisaged.

[0039] The present invention may preferably be integrated with the rulesset forth for zinc finger polypeptide engineering in our copendingEuropean or PCT patent applications having publication numbers WO98/53057, WO 98/53060, WO 98/53058, which describe improved techniquesfor designing zinc finger polypeptides capable of binding desirednucleic acid sequences. In combination with selection procedures, suchas phage display, set forth for example in WO 96/06166, these techniquesenable the production of zinc finger polypeptides capable of recognisingpractically any desired sequence.

[0040] A zinc finger binding motif is a structure well known to those inthe art and defined in, for example, Miller et al., (1985) EMBO J.4:1609-1614; Berg (1988) PNAS (USA) 85:99-102; Lee et al., (1989)Science 245:635-637; see International patent applications WO 96/06166and WO 96/32475, corresponding to U.S. Ser. No. 08/422,107, incorporatedherein by reference.

[0041] In general, a preferred zinc finger framework has the structure:

[0042] (A) X0-2C X1-5C X9-14H X3-6H/C

[0043] where X is any amino acid, and the numbers in subscript indicatethe possible numbers of residues represented by X.

[0044] In a preferred aspect of the present invention, zinc fingernucleic acid binding motifs may be represented as motifs having thefollowing primary structure:

[0045] (B) Xa C X2-4C X2-3F Xc X X X X L X X H X X Xb H-linker

[0046] −1 1 2 3 4 5 6 7 8 9

[0047] wherein X (including X^(a), X^(b) and X^(c)) is any amino acid.X₂₋₄ and X₂₋₃ refer to the presence of 2 or 4, or 2 or 3, amino acids,respectively. The Cys and His residues, which together co-ordinate thezinc metal atom, are marked in bold text and are usually invariant, asis the Leu residue at position +4 in the α-helix.

[0048] Modifications to this representation may occur or be effectedwithout necessarily abolishing zinc finger function, by insertion,mutation or deletion of amino acids. For example it is known that thesecond His residue may be replaced by Cys (Krizek el al., (1991) J. Am.Chem. Soc. 113:4518-4523) and that Leu at +4 can in some circumstancesbe replaced with Arg. The Phe residue before X_(c) may be replaced byany aromatic other than Trp. Moreover, experiments have shown thatdeparture from the preferred structure and residue assignments for thezinc finger are tolerated and may even prove beneficial in binding tocertain nucleic acid sequences. Even taking this into account, however,the general structure involving an α-helix co-ordinated by a zinc atomwhich contacts four Cys or His residues, does not alter. As used herein,structures (A) and (B) above are taken as an exemplary structurerepresenting all zinc finger structures of the Cys2-His2 type.

[0049] Preferably, X^(a) is ^(F)/_(Y)-X or P-^(F)/_(Y)-X. In thiscontext, X is any amino acid. Preferably, in this context X is E, K, Tor S. Less preferred but also envisaged are Q, V, A and P. The remainingamino acids remain possible.

[0050] Preferably, X₂₋₄ consists of two amino acids rather than four.The first of these amino acids may be any amino acid, but S, E, K, T, Pand R are preferred. Advantageously, it is P or R. The second of theseamino acids is preferably E, although any amino acid may be used.

[0051] Preferably, X^(b) is T or 1. Preferably, X^(c) is S or T.

[0052] Preferably, X₂₋₃ is G-K-A, G-K-C, G-K-S or G-K-G. However,departures from the preferred residues are possible, for example in theform of M-R-N or M-R.

[0053] Preferably, the linker is T-G-E-K or T-G-E-K-P.

[0054] As set out above, the major binding interactions occur with aminoacids −1, +3 and +6. Amino acids +4 and +7 are largely invariant. Theremaining amino acids may be essentially any amino acids. Preferably,position +9 is occupied by Arg or Lys. Advantageously, positions +1, +5and +8 are not hydrophobic amino acids, that is to say are not Phe, Trpor Tyr. Preferably, position ++2 is any amino acid, and preferablyserine, save where its nature is dictated by its role as a ++2 aminoacid for an N-terminal zinc finger in the same nucleic acid bindingmolecule.

[0055] In a most preferred aspect, therefore, bringing together theabove, the invention allows the definition of every residue in a zincfinger DNA binding motif which will bind specifically to a given targetDNA triplet.

[0056] The code provided by the present invention is not entirely rigid;certain choices are provided. For example, positions +1, +5 and +8 mayhave any amino acid allocation, whilst other positions may have certainoptions: for example, the present rules provide that, for binding to acentral T residue, any one of Ala, Ser or Val may be used at +3. In itsbroadest sense, therefore, the present invention provides a very largenumber of proteins which are capable of binding to every defined targetDNA triplet.

[0057] Preferably, however, the number of possibilities may besignificantly reduced. For example, the non-critical residues +1, +5 and+8 may be occupied by the residues Lys, Thr and Gln respectively as adefault option. In the case of the other choices, for example, thefirst-given option may be employed as a default. Thus, the codeaccording to the present invention allows the design of a single,defined polypeptide (a “default” polypeptide) which will bind to itstarget triplet.

[0058] Accordingly, the zinc fingers of the present invention can beprepared using a method comprising the steps of: (a) selecting a modelzinc finger domain from the group consisting of naturally occurring zincfingers and consensus zinc fingers; and (b) mutating at least one ofpositions −1, +3, +6 (and ++2) of the finger.

[0059] In general, naturally occurring zinc fingers may be selected fromthose fingers for which the DNA binding specificity is known. Forexample, these may be the fingers for which a crystal structure has beenresolved: namely Zif 268 (Elrod-Erickson et al., (1996) Structure4:1171-1180), GLI (Pavletich and Pabo, (1993) Science 261:1701-1707),Tramtrack (Fairall et al., (1993) Nature 366:483-487) and YY1 (Houbaviyet al., (1996) PNAS (USA) 93:13577-13582).

[0060] Although mutation of the DNA-contacting amino acids of the DNAbinding domain allows selection of polypeptides which bind to desiredtarget nucleic acids, in a preferred embodiment residues which areoutside the DNA-contacting region may be mutated. Mutations in suchresidues may affect the interaction between zinc fingers in a zincfinger polypeptide, and thus alter binding site specificity.

[0061] The naturally occurring zinc finger 2 in Zif268 makes anexcellent starting point from which to engineer a zinc finger and ispreferred.

[0062] Consensus zinc finger structures may be prepared by comparing thesequences of known zinc fingers, irrespective of whether their bindingdomain is known. Preferably, the consensus structure is selected fromthe group consisting of the consensus structure P Y K C P E C G K S F SQ K S D L V K H Q R T H T G, and the consensus structure P Y K C S E C GK A F S Q K S N L T R H Q R I H T G E K P.

[0063] The consensuses are derived from the consensus provided by Krizekel al., (1991) J. Am. Chem. Soc. 113: 4518-4523 and from Jacobs, (1993)PhD thesis, University of Cambridge, UK. In both cases, the linkersequences described above for joining two zinc finger motifs together,namely TGEK or TGEKP can be formed on the ends of the consensus. Thus, aP may be removed where necessary, or, in the case of the consensusterminating T G, E K (P) can be added.

[0064] The present invention provides methods of engineering and usingzinc finger proteins in plants which zinc finger proteins are capable ofbinding to a target DNA sequence, and wherein binding to each base ofthe triplet by an α-helical zinc finger DNA binding motif in thepolypeptide is determined as follows:

[0065] (a) if the 5′ base in the triplet is G, then position +6 in theα-helix is Arg and/or position ++2 is Asp;

[0066] (b) if the 5′ base in the triplet is A, then position +6 in theα-helix is Gln or Glu and ++2 is not Asp;

[0067] (c) if the 5′ base in the triplet is T, then position +6 in theα-helix is Ser or Thr and position ++2 is Asp; or position +6 is ahydrophobic amino acid other than Ala;

[0068] (d) if the 5′ base in the triplet is C, then position +6 in theα-helix may be any amino acid, provided that position ++2 in the α-helixis not Asp;

[0069] (e) if the central base in the triplet is G, then position +3 inthe α-helix is His;

[0070] (f) if the central base in the triplet is A, then position +3 inthe α-helix is Asn;

[0071] (g) if the central base in the triplet is T, then position +3 inthe α-helix is Ala, Ser, Ile, Leu, Thr or Val; provided that if it isAla, then one of the residues at −1 or +6 is a small residue;

[0072] (h) if the central base in the triplet is 5-meC, then position +3in the α-helix is Ala, Ser, Ile, Leu, Thr or Val; provided that if it isAla, then one of the residues at −1 or +6 is a small residue;

[0073] (i) if the 3′ base in the triplet is G, then position −1 in theα-helix is Arg;

[0074] (j) if the 3′ base in the triplet is A, then position −1 in theα-helix is Gin and position +2 is Ala;

[0075] (k) if the 3′ base in the triplet is T, then position −1 in theα-helix is Asn; or position −1 is Gin and position +2 is Ser;

[0076] (l) if the 3′ base in the triplet is C, then position −1 in theα-helix is Asp and Position +1 is Arg; where the central residue of atarget triplet is C, the use of Asp at position +3 of a zinc fingerpolypeptide allows preferential binding to C over 5-meC.

[0077] The foregoing represents a set of rules which permits the designof a zinc finger binding protein specific for any given target DNAsequence.

[0078] When the nucleic acid specificity of the model finger selected isknown, the mutation of the finger in order to modify its specificity tobind to the target DNA may be directed to residues known to affectbinding to bases at which the natural and desired targets differ.Otherwise, mutation of the model fingers should be concentrated uponresidues −1, +3, +6 and ++2 as provided for in the foregoing rules.

[0079] In order to produce a binding protein having improved binding,moreover, the rules provided by the present invention may besupplemented by physical or virtual modelling of the protein/DNAinterface in order to assist in residue selection.

[0080] Methods for the production of libraries encoding randomisedpolypeptides are known in the art and may be applied in the presentinvention. Randomisation may be total, or partial; in the case ofpartial randomisation, the selected codons preferably encode options foramino acids as set forth in the rules above.

[0081] The invention encompasses library technology described in ourcopending International patent application WO 98/53057, incorporatedherein by reference in its entirety. WO 98/53057 describes theproduction of zinc finger polypeptide libraries in which each individualzinc finger polypeptide comprises more than one, for example two orthree, zinc fingers; and wherein within each polypeptide partialrandomisation occurs in at least two zinc fingers.

[0082] This allows for the selection of the “overlap” specificity,wherein, within each triplet, the choice of residue for binding to thethird nucleotide (read 3′ to 5′ on the + strand) is influenced by theresidue present at position +2 on the subsequent zinc finger, whichdisplays cross-strand specificity in binding. The selection of zincfinger polypeptides incorporating cross-strand specificity of adjacentzinc fingers enables the selection of nucleic acid binding proteins morequickly, and/or with a higher degree of specificity than is otherwisepossible.

[0083] Zinc finger binding motifs engineered for use in accordance withthe present invention may be combined into nucleic acid bindingpolypeptide molecules having a multiplicity of zinc fingers. Preferably,the proteins have at least two zinc fingers. In nature, zinc fingerbinding proteins commonly have at least three zinc fingers, althoughtwo-zinc finger proteins such as Tramtrack are known. The presence of atleast three zinc fingers is preferred. Nucleic acid binding proteins maybe constructed by joining the required fingers end to end, N-terminus toC-terminus. Preferably, this is effected by joining together therelevant nucleic acid sequences which encode the zinc fingers to producea composite nucleic acid coding sequence encoding the entire bindingprotein.

[0084] A “leader” peptide may be added to the N-terminal finger.Preferably, the leader peptide is MAEEKP.

[0085] Zinc finger polypeptides comprising more than three zinc fingers,such as four, five, six, seven, eight or nine zinc fingers can also beused in conjunction with the present invention. Linkers that arepreferably used to link zinc finger domains are described in co-pendingpatent applications GB 0013102.9, GB 0013103.7and GB 0013104.5 filed onMay, 30, 2000. An example of a multiple zinc finger protein described inthis specification comprises zinc fingers 1-3 of TFIIIA and the threezinc fingers from Zif268 joined by zinc finger 4, including flankingsequences, of TFIIIA. We have called the zinc finger protein TFIIIAZif.Zinc finger 4 of TFIIIA does not bind DNA but acts as a linker inbetween the two sets of zinc fingers that are involved in DNArecognition. Despite the fact that this zinc finger does not make anybase contacts within the major groove of the DNA, it is folded in theclassical way, for Cys2His2 zinc fingers, around a Zn(II) ion and isfolded to contain an alpha helix within its structure (Nolte et al.,1998). However, other linkers can be used in conjunction with thepresent invention to construct zinc finger proteins comprising multiplezinc finger domains.

[0086] B. Target Genes

[0087] Examples of target genes include any gene involved in any traitthat may be of interest to a farmer or grower, a processor, or aconsumer of a plant or plant product.

[0088] For example, genes involved in the starch characteristics areuseful target genes and the present invention can be used in conjunctionwith starch branching enzyme (for example) to generate corn plants whichgenerate seed with super-branching starch.

[0089] Genes involved in oil characteristics are useful target genes andthe present invention can be used in conjunction withdelta-12-desaturase (for example) to generate corn plants which generateseed with higher oleic and lower linoleic acid.

[0090] Genes involved in cotton fiber characteristics are useful targetgenes and the technology of the present invention can be used to modifythe expression of such genes to improve traits such as fiber strength.

[0091] A further example is provided by the genes involved in thebiosynthesis and catabolism of gibberellins. The gibberellins are aclass of plant hormones involved in the determination of many planttraits including elongation growth, bolting/flowering, leaf expansion,seed set, fruit size and dormancy. Accordingly, the regulation of genesinvolved in the biosynthesis and catabolism of gibberellins can be usedto generate plants such as wheat, corn, sugar beet and sugar cane withimproved traits (Phillips et al. (1995) Plant Physiol. 108:1049-1057;MacMillin et al. (1997) Plant Physiol. 113:1369-1377; Williams et al.(1998) Plant Physiol. 117:559-563; and Thomas et al. (1999) Proc. Natl.Acad. Sci. USA 96:4698-4703).

[0092] Genes involved in nitrogen metabolism (for example glutaminesynthetase, asparagine synthetase, GOGAT, glutamate dehydrogenase) areuseful target genes and the technology of the present invention can beused to modify the expression of such genes to improve nitrogen useefficiency in plants and thereby to reduce the requirement for theapplication of inorganic fertilizers.

[0093] Genes involved in the biosynthesis of plant cell components suchas cellulose and lignin can be targeted by the technology of the presentinvention to modify digestibility in crops such as corn which are usedas silage.

[0094] Genes whose products are responsible for ripening (such aspolygalacturonase and ACC oxidase) are interesting target genes as thepresent invention can be used to modify ripening characteristics inplants such as tomato, avocado, and banana.

[0095] Genes involved in the biosynthesis of volatile esters, which areimportant flavor compounds in fruits and vegetables, are equallyinteresting target genes and the present invention can be used toimprove such traits (Dudavera et al. (1996) Plant Cell 8:1137-1148;Dudavera et al. (1998) Plant J. 14:297-304; Ross et al. (1999) Arch.Biochem. Biophys. 367:9-16).

[0096] Genes which are involved in the biosynthesis of plant-derivedpharmaceutically important compounds are also potential target gene.Using the technology of the present invention, the up-regulation of arate-limiting step in the biosynthetic pathway of a pharmaceuticallyimportant compound will result in the production of higher levels ofsuch compound in the plant.

[0097] Additionally, target genes used in conjunction with the presentinvention include genes encoding allergens such as the peanut allergensArah1, Arah2 and Arah3. Rabjohn et al. J. Clin. Invest. 103:535-542.Down-regulation of such genes using the technology of the presentinvention is expected to reduce the allergenicity of the transgenicpeanuts.

[0098] Examples of heterologous target genes include genes which areintroduced into a plant for the production of biodegradable plastic (forexample) but which are placed under the regulatory control of a zincfinger protein of the present invention.

[0099] C. Target DNA Sequence

[0100] Most commonly, target DNA sequences will be sequences associatedwith a target gene that is to be regulated by a zinc finger protein ofthe present invention. Target DNA sequences include note only sequenceswhich are naturally associated with target genes, but also othersequences which can be configured with a gene of interest to allow theup- or down regulation of such a gene of interest. For example, theknown binding site of a given zinc finger protein can be a targetsequence and, when operably linked to a gene of interest, will allow thegene of interest to be regulated by the given zinc finger protein.

[0101] D. DNA Libraries

[0102] DNA sequences for use in screening methods to select zinc fingersand corresponding DNA sequences can be provided as a library of relatedsequences having homology to one another (as opposed to a genomiclibrary, for example, obtained by cutting up a large amount ofessentially unrelated sequences).

[0103] A library of DNA sequences can be used in at least two differentways. First, it can be used in a screen to identify zinc fingers thatbind to a specific sequence. Second, it can be used to confirm thespecificity of selected zinc fingers.

[0104] A DNA library is advantageously used to test the selectivity of azinc finger for nucleotide sequences of length N. Consequently, sincethere are four different nucleotides that occur naturally in genomicDNA, the total number of sequences required to represent all possiblebase permutations for a sequence of length N is 4^(N). N is an integerhaving a value of at least three. That it to say that the smallestlibrary envisaged for testing binding to a nucleotide sequence whereonly one DNA triplet is varied, consists of 64 different sequences.However, N can be any integer greater than or equal to 3 such as 4, 5,6, 7, 8 or 9. Typically, N only needs to be three times the number ofzinc fingers being tested, optionally included a few additional residuesoutside of the binding site that can influence specificity. Thus, by wayof example, to test the specificity of a protein comprising three zincfingers, where all three fingers have been engineered, it can bedesirable to use a library where N is at least 9.

[0105] Libraries of DNA sequences can be screened using a number ofdifferent methods. For example, the DNA can be immobilised to beads andincubated with zinc fingers that are labelled with an affinity ligandsuch as biotin or expressed on the surface of phage. Complexes betweenthe DNA and zinc finger can be selected by washing the beads to removeunbound zinc fingers and then purifying the beads using the affinityligand bound to the zinc fingers to remove beads that do not containbound zinc fingers. Any remaining beads should contain DNA/zinc fingercomplexes. Individual beads can be selected and the identity of the DNAand zinc finger determined. Other modifications to the technique includethe use of detectable labels, for example fluorescently labelling thezinc fingers and sorting beads that have zinc fingers bound to them byFACS.

[0106] In an alternative method, the DNA sequences in the library areimmobilised at discrete positions on a solid substrate, such as a DNAchip, such that each different sequence is separated from othersequences on the solid substrate. Binding of zinc finger proteins isdetermined as described below and individual proteins isolated (whichcan be conveniently achieved by the use of phage display techniques).This technique can also be used as a second step after a zinc finger hasbeen selected by, for example, the bead method described above, tocharacterise fully the binding specificity of a selected zinc finger

[0107] In a DNA library, it is generally not necessary or desirable forall positions to be randomised. Preferably only a subsequence of N basesof the complete DNA sequence is varied. The 4^(N) possible permutationsof the DNA sequence of length N sequence are typically arranged in 4Nsub-libraries, wherein for any one sub-library one base in the DNAsequence of length N is defined and the other N-1 bases are randomised.Thus in the case of a varied DNA triplet, there will be 12sub-libraries.

[0108] As mentioned above, the nucleotide sequence of length N isgenerally part of a longer DNA molecule. However, the nucleotidesequence of length N typically occupies the same position within thelonger molecule in each of the varied sequences even though the sequenceof N itself can vary. The other sequences within the DNA molecule aregenerally the same throughout the library. Thus the library can be saidto consist of a library of 4^(N) DNA molecules of the formulaR¹-(A/C/G/T)₄ ^(N)-R², wherein R¹ and R² can be any nucleotide sequence.

[0109] Preferably, each sequence is also represented as adilution/concentration series. Thus the immobilized DNA library canoccupy Z4^(N) discrete positions on the chip where Z is the number ofdifferent dilutions in the series and is an integer having a value of atleast 2. The range of DNA concentrations for the dilution series istypically in the order of 0.01 to 100 pmol cm⁻², preferably from 0.05 to5 pmol cm⁻². The concentrations typically vary 10-fold, i.e. a seriescan consist of 0.01, 0.1, 1, 10 and 100 pmol cm⁻², but can vary, forexample, by 2- or 5-fold.

[0110] The advantage of including the DNA sequences in a dilution seriesis that it is then possible to estimate K_(d)s for protein/DNA complexesusing standard techniques such as the Kaleidagraph™ version 2.0 program(Abelback Software).

[0111] The DNA molecules in the library are at least partiallydouble-stranded, in particular at least the nucleotide sequence oflength N is double-stranded. Single stranded regions can be included,for example to assist in attaching the DNA library to the solidsubstrate.

[0112] Techniques for producing immobilized libraries of DNA moleculeshave been described in the art. Generally, most prior art methodsdescribed how to synthesize single-stranded nucleic acid moleculelibraries, using for example masking techniques to build up variouspermutations of sequences at the various discrete positions on the solidsubstrate. U.S. Pat. No. 5,837,832 (the '832 patent), describes animproved method for producing DNA arrays immobilized to siliconsubstrates based on very large scale integration technology. Inparticular, the '832 patent describes a strategy called “tiling” tosynthesize specific sets of probes at spatially-defined locations on asubstrate which can be used to produced the immobilized DNA libraries ofthe present invention. The '832 patent also provides references forearlier techniques that can also be used.

[0113] However, an important aspect of the present invention is that itrelates to DNA binding proteins, zinc fingers that bind double-strandedDNA. Thus single-stranded nucleic acid molecule libraries using theprior art techniques referred to above will then need to be converted todouble-stranded DNA libraries by synthesizing a complementary strand. Anexample of the conversion of single-stranded nucleic acid moleculelibraries to double-stranded DNA libraries is given in Bulyk el al.(1999) Nature Biotechnol. 17:573-577. The technique described in Bulyket al. (1999) typically requires the inclusion of a constant sequence inevery member of the library (i.e. within R¹ or R² in the generic formulagiven above) to which a nucleotide primer is bound to act as a primerfor second strand synthesis using a DNA polymerase and other appropriatereagents. If required, deoxynucleotide triphosphates (dNTPs) having adetectable labeled can be included to allow the efficiency of secondstrand synthesis to be monitored. Also the detectable label can assistin detecting binding of zinc fingers when the immobilized DNA library isin use.

[0114] Alternatively, double-stranded molecules can be synthesized offthe solid substrate and each pre-formed sequence applied to a discreteposition on the solid substrate. An example of such a method is tosynthesis palindromic single-stranded nucleic acids. See U.S. Pat. No.5,556,752.

[0115] Thus DNA can typically be synthesized in situ on the surface ofthe substrate. However, DNA can also be printed directly onto thesubstrate using for example robotic devices equipped with either pins orpiezo electric devices.

[0116] The library sequences are typically immobilized onto or indiscrete regions of a solid substrate. The substrate can be porous toallow immobilization within the substrate or substantially non-porous,in which case the library sequences are typically immobilized on thesurface of the substrate. The solid substrate can be made of anymaterial to which polypeptides can bind, either directly or indirectly.Examples of suitable solid substrates include flat glass, siliconwafers, mica, ceramics and organic polymers such as plastics, includingpolystyrene and polymethacrylate. It can also be possible to usesemi-permeable membranes such as nitrocellulose or nylon membranes,which are widely available. The semi-permeable membranes can be mountedon a more robust solid surface such as glass. The surfaces canoptionally be coated with a layer of metal, such as gold, platinum orother transition metal. A particular example of a suitable solidsubstrate is the commercially available BiaCore™ chip (PharmaciaBiosensors).

[0117] Preferably, the solid substrate is generally a material having arigid or semi-rigid surface. In preferred embodiments, at least onesurface of the substrate will be substantially flat, although in someembodiments it can be desirable to physically separate synthesis regionsfor different polymers with, for example, raised regions or etchedtrenches. Preferably the solid substrate is not a microtiter plate orbead. It is also preferred that the solid substrate is suitable for thehigh density application of DNA sequences in discrete areas of typicallyfrom 50 to 100 μm, giving a density of 10000 to 40000 cm⁻².

[0118] The solid substrate is conveniently divided up into sections.This can be achieved by techniques such as photoetching, or by theapplication of hydrophobic inks, for example Teflon-based inks(Cel-line, USA).

[0119] Discrete positions, in which each different member of the libraryis located can have any convenient shape, e.g., circular, rectangular,elliptical, wedge-shaped, etc.

[0120] Attachment of the library sequences to the substrate can be bycovalent or non-covalent means. The library sequences can be attached tothe substrate via a layer of molecules to which the library sequencesbind. For example, the library sequences can be labeled with biotin andthe substrate coated with avidin and/or streptavidin. A convenientfeature of using biotinylated library sequences is that the efficiencyof coupling to the solid substrate can be determined easily. Since thelibrary sequences can bind only poorly to some solid substrates, it isoften necessary to provide a chemical interface between the solidsubstrate (such as in the case of glass) and the library sequences.

[0121] Examples of suitable chemical interfaces include hexaethyleneglycol. Another example is the use of polylysine coated glass, thepolylysine then being chemically modified using standard procedures tointroduce an affinity ligand. Other methods for attaching molecules tothe surfaces of solid substrate by the use of coupling agents are knownin the art, see for example WO98/49557.

[0122] Binding of zinc fingers to the immobilized DNA library can bedetermined by a variety of means such as changes in the opticalcharacteristics of the bound DNA (i.e. by the use of ethidium bromide)or by the use of labeled zinc finger polypeptides, such as epitopetagged zinc finger polypeptides or zinc finger polypeptides labeled withfluorophores such as green fluorescent protein (GFP). Other detectiontechniques that do not require the use of labels include opticaltechniques such as optoacoustics, reflectometry, ellipsometry andsurface plasma resonance (SPR). See, WO97/49989.

[0123] Binding of epitope tagged zinc finger polypeptides is typicallyassessed by immunological detection techniques where the primary orsecondary antibody comprises a detectable label. A preferred detectablelabel is one that emits light, such as a fluorophore, for examplephycoerythrin.

[0124] The complete DNA library is typically read at the same time bycharged coupled device (CCD) camera or confocal imaging system.Alternatively, the DNA library can be placed for detection in a suitableapparatus that can move in an X-Y direction, such as a plate reader. Inthis way, the change in characteristics for each discrete position canbe measured automatically by computer controlled movement of the arrayto place each discrete element in turn in line with the detection means.

[0125] E. Nucleic Acid Vectors Encoding Zinc Finger Proteins

[0126] Polynucleotides encoding zinc finger proteins for use in theinvention can be incorporated into a recombinant replicable vector. Thevector can be used to replicate the nucleic acid in a compatible hostcell and the vector can be recovered from the host cell. Suitable hostcells include bacteria such as Escherichia coli, yeast and eukaryoticcell lines.

[0127] Preferably, a polynucleotide encoding a zinc finger proteinaccording to the invention in a vector is operably linked to a controlsequence that is capable of providing for the expression of the codingsequence by the host cell, i.e. the vector is an expression vector. Theterm “operably linked” means that the components described are in arelationship permitting them to function in their intended manner. Aregulatory sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved undercondition compatible with the control sequences.

[0128] The control sequences can be modified, for example by theaddition of further transcriptional regulatory elements to make thelevel of transcription directed by the control sequences more responsiveto transcriptional modulators.

[0129] Vectors of the invention can be transformed or transfected into asuitable host cell as described below to provide for expression of aprotein of the invention.

[0130] The vectors can be for example, plasmid or virus vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.The vectors can contain one or more selectable marker genes and thesewill vary depending on the system used, but are known to those of skillin the art. Vectors can be used, for example, to transfect or transforma host cell.

[0131] Control sequences operably linked to sequences encoding theprotein of the invention include promoters/enhancers and otherexpression regulation signals such as terminators. These controlsequences can be selected to be compatible with the host cell in whichthe expression vector is designed to be used. The term promoter iswell-known in the art and encompasses nucleic acid regions ranging insize and complexity from minimal promoters to promoters includingupstream elements and enhancers.

[0132] The promoter is typically selected from promoters which arefunctional in plant cells, although prokaryotic promoters and promotersfunctional in other eukaryotic cells can be used.

[0133] Typically, the promoter is derived from viral or plant genesequences. For example, the promoter can be derived from the genome of acell in which expression is to occur. With respect to plant promoters,they can be promoters that function in a ubiquitous manner or,alternatively, a tissue-specific manner. Tissue-specific promotersspecific for different tissues of the plant are particularly preferred.Examples are provided below. Tissue-specific expression can be used toconfine expression of the binding domain and/or binding partner to acell type or tissue/organ of interest. Promoters can also be used thatrespond to specific stimuli, for example promoters that are responsiveto plant hormones. Viral promoters can also be used, for example theCaMV 35S promoter well known in the art.

[0134] It can also be advantageous for the promoters to be inducible sothat the levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated. Inducibleexpression allows the researcher to control when expression of thepolypeptides takes place.

[0135] In addition, any of these promoters can be modified by theaddition of further regulatory sequences, for example enhancersequences. Chimeric promoters can also be used comprising sequenceelements from two or more different promoters described above.

[0136] Advantageously, a plant expression vector encoding a zinc fingerprotein according to the invention can comprise a locus control region(LCR). LCRs are capable of directing high-level integration siteindependent expression of transgenes integrated into host cell chromatin(Stief et al. (1989) Nature 341:343); Lang et al. (1991) Nucl. AcidsRes. 19:5851-5856.).

[0137] According to the invention, the zinc finger protein constructs ofthe invention are expressed in plant cells under the control oftranscriptional regulatory sequences that are known to function inplants. The regulatory sequences selected will depend on the requiredtemporal and spatial expression pattern of the zinc finger protein inthe host plant. Many plant promoters have been characterized and wouldbe suitable for use in conjunction with the invention. By way ofillustration, some examples are provided below:

[0138] A large number of promoters are known in the art which directexpression in specific tissues and organs (e.g. roots, leaves, flowers)or in cell types (e.g. leaf epidermal cells, leaf mesophyll cells, rootcortex cells). For example, the maize PEPC promoter from the phosphoenolcarboxylase gene (Hudspeth and Grula (1989) Plant Mol. Biol. 12:579-589)is green tissue-specific; the trpA gene promoter is pith cell-specific(WO 93/07278 to Ciba-Geigy); the TA29 promoter is pollen-specific.Mariani et al. (1990) Nature 347:737-741; and Mariani et al. (1992)Nature 357:384-387.

[0139] Other promoters direct transcription under conditions of presenceof light or absence or light or in a circadian manner. For example, theGS2 promoter described by Edwards and Coruzzi (1989) Plant Cell1:241-248 is induced by light, whereas the AS1 promoter described byTsai and Coruzzi (1990) EMBO J. 9:323-332 is expressed only inconditions of darkness.

[0140] Other promoters are wound-inducible and typically directtranscription not just on wound induction, but also at the sites ofpathogen infection. Examples are described by Xu et al. (1993) PlantMol. Biol. 22:573-588; Logemann et al. (1989) Plant Cell 1:151-158; andFirek et al. (1993) Plant Mol. Biol. 22:129-142.

[0141] Further plant promoters of interest are the bronze promoter(Ralston et al. (1988) Genetics 119:185-197 and Genbank Accession No.X07937.1) which directs expression of UDPglucose flavanoidglycosyl-transferase in maize, the patatin-1 gene promoter (Jefferson etal. (1990) Plant Mol. Biol. 14:995-1006) that contains sequences capableof directing tuber-specific expression, and the phenylalanine ammonialyase promoter (Bevan et al. (1989) EMBO J. 8:1899-1906) though to beinvolved in responses to mechanical wounding and normal development ofthe xylem and flower.

[0142] A number of constitutive promoters can be used in plants. Theseinclude the Cauliflower Mosaic Virus (CaMV) 35S promoter (U.S. Pat. No.5,352,605 and U.S. Pat. No. 5,322,938, both to Monsanto) includingminimal promoters (such as the −90 CaMV 35S promoter) linked to otherregulatory sequences, the rice actin promoter (McElroy et al. (1991)Mol. Gen. Genet. 231:150-160), and the maize and sunflower ubiquitinpromoters. Christensen et al. (1989) Plant Mol. Biol. 12:619-632; andBinet et al. (1991) Plant Science 79:87-94).

[0143] A further promoter of interest is the inducible promoterdescribed by Aoyama and Chua (1997) Plant J. 11:605-612; and Zou andChua (2000) Curr. Op. Biotech. 11: 146-151. By using this induciblepromoter system, transgenic lines can be established which carry thezinc finger chimera but express it only after addition of an inducer..Thus the zinc fingers of the present invention can be expressed inresponse to the inducer allowing the dose or level of zinc fingerprotein in the cell or plant to the adjusted to a desired amount.

[0144] Using promoters that direct transcription in the plant species ofinterest, the zinc finger protein of the invention can be expressed inthe required cell or tissue types. For example, if it is the intentionto utilize the zinc finger protein to regulate a gene in a specific cellor tissue type, then the appropriate promoter can be used to directexpression of the zinc finger protein construct.

[0145] An appropriate terminator of transcription is fused downstream ofthe selected zinc finger protein containing transgene and any of anumber of available terminators can be used in conjunction with theinvention. Examples of transcriptional terminator sequences that areknown to function in plants include the nopaline synthase terminatorfound in the pBI vectors (Clontech catalog 1993/1994), the E9 terminatorfrom the rbcS gene, and the tml terminator from CaMV.

[0146] A number of sequences found within the transcriptional unit areknown to enhance gene expression and these can be used within thecontext of the current invention. Such sequences include intronsequences which, particularly in monocotyledonous cells, are known toenhance expression. Both intron 1 of the maize Adh1 gene and the intronfrom the maize bronze 1 gene have been found to be effective inenhancing expression in maize cells (Callis et al. (1987) Genes Develop.1:1183-1200) and intron sequences are frequently incorporated into planttransformation vectors, typically within the non-translated leader.

[0147] A number of virus-derived non-translated leader sequences havebeen found to enhance expression, especially in dicotyledonous cells.Examples include the “Ω” leader sequence of Tobacco Mosaic Virus, andsimilar leader sequences of Maize Chlorotic Mottle Virus and AlfalfaMosaic Virus. Gallie et al. (1987) Nucl. Acids Res. 15:8693-8711; andShuzeski et al. (1990) Plant Mol. Biol. 15:65-79.

[0148] The zinc finger proteins of the current invention are targeted tothe cell nucleus so that they are able to interact with host cell DNAand bind to the appropriate DNA target in the nucleus and regulatetranscription. To effect this, a Nuclear Localization Sequence (NLS) isincorporated in frame with the expressible zinc finger construct. TheNLS can be fused either 5′ or 3′ to the zinc finger encoding sequence.

[0149] The NLS of the wild-type Simian Virus 40 Large T-Antigen(Kalderon et al. (1984) Cell 37:801-813; and Markland et al. (1987) Mol.Cell. Biol. 7:4255-4265) is an appropriate NLS and has previously beenshown to provide an effective nuclear localization mechanism in plants.van der Krol et al. (1991) Plant Cell 3:667-675. However, severalalternative NLSs are known in the art and can be used instead of theSV40 NLS sequence. These include the Nuclear Localization Signals ofTGA-1A and TGA-1B (van der Krol et al. (1991)).

[0150] A variety of transformation vectors are available for planttransformation and the zinc finger protein encoding genes of theinvention can be used in conjunction with any such vectors. Theselection of vector will depend on the preferred transformationtechnique and the plant species that is to be transformed. For certaintarget species, different selectable markers can be preferred.

[0151] For Agrobacterium-mediated transformation, binary vectors orvectors carrying at least one T-DNA border sequence are suitable. Anumber of vectors are available including pBIN19 (Bevan (1984) Nucl.Acids Res. 12:8711-8721), the pBI series of vectors, and pCIB10 andderivatives thereof. Rothstein et al. (1987) Gene 53:153-161; and WO95/33818.

[0152] Binary vector constructs prepared for Agrobacieriumtransformation are introduced into an appropriate strain ofAgrobacterium tumefaciens (for example, LBA 4044 or GV 3101) either bytriparental mating or direct transformation. Bevan (1984); and Hofgenand Willmitzer, Nucl. Acids Res. 16:9877 (1988).

[0153] For transformation which is not Agrobacterium-mediated (i.e.direct gene transfer), any vector is suitable and linear DNA containingonly the construct of interest can be preferred. Direct gene transfercan be undertaken using a single DNA species or multiple DNA species(cotransformation; Schroder et al. (1986) Biotechnol. 4:1093-1096).

[0154] Construction of vectors according to the invention employsconventional ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required. If desired, analysis to confirm correct sequences inthe constructed plasmids is performed in a known fashion. Suitablemethods for constructing expression vectors, preparing in vitrotranscripts, introducing DNA into host cells, and performing analysesfor assessing DNA binding protein expression and function are known tothose skilled in the art. Gene presence, amplification and/or expressioncan be measured in a sample directly, for example, by conventionalSouthern blotting, Northern blotting to quantitate the transcription ofmRNA, dot blotting (DNA or RNA analysis), or in situ hybridization,using an appropriately labeled probe which can be based on a sequenceprovided herein. Those skilled in the art will readily envisage howthese methods can be modified, if desired.

[0155] DNA can be stably incorporated into cells or can be transientlyexpressed using methods known in the art. Stably transfected cells canbe prepared by transfecting cells with an expression vector having aselectable marker gene, and growing the transfected cells underconditions selective for cells expressing the marker gene. To preparetransient transfectants, cells are transfected with a reporter gene tomonitor transfection efficiency.

[0156] Heterologous DNA can be introduced into plant host cells by anymethod known in the art, such as electroporation or A. tumefaciensmediated transfer. Although specific protocols can vary from species tospecies, transformation techniques are well known in the art for mostcommercial plant species.

[0157] In the case of dicotyledonous species, Agrobacterium-mediatedtransformation is generally a preferred technique as it has broadapplication to many dicotyledons species and is generally veryefficient. Agrobacterium-mediated transformation generally involves theco-cultivation of Agrobacterium with explants from the plant and followsprocedures and protocols that are known in the art. Transformed tissueis generally regenerated on medium carrying the appropriate selectablemarker. Protocols are known in the art for many dicotyledonous cropsincluding (for example) cotton, tomato, canola and oilseed rape, poplar,potato, sunflower, tobacco and soybean (see for example EP 0 317 511, EP0 249 432, WO 87/07299, U.S. Pat. No. 5,795,855).

[0158] In addition to Agrobacterium-mediated transformation, variousother techniques can be applied to dicotyledons. These includepolyethylene glycol (PEG) and electroporation-mediated transformation ofprotoplasts, and microinjection. Potrykus et al. (1985) Mol. Gen. Genet.199:169-177; Reich et al. (1986) Biotechnol. 4:1001-1004; Klein et al.(1987) Nature 327:70-73. As with Agrobacterium-mediated transformation,transformed tissue is generally regenerated on medium carrying theappropriate selectable marker using standard techniques known in theart.

[0159] Although Agrobacterium-mediated transformation has been appliedsuccessfully to monocotyledonous species such as rice and maize andprotocols for these approaches are available in the art, the most widelyused transformation techniques for monocotyledons remain particlebombardment, and PEG and electroporation-mediated transformation ofprotoplasts.

[0160] In the case of maize, techniques are available for transformationusing particle bombardment. Gordon-Kamm et al. (1990) Plant Cell2:603-618; Fromm et al. (1990) Biotechnol. 8:833-839; and Koziel et al.(1993) Biotechnol. 11:194-200. The preferred method is the use ofbiolistics using, for instance, gold or tungsten. Suitable methods areknown in the art and described, for instance, in U.S. Pat. Nos.5,489,520 and 5,550,318. See also, Potrykus (1990) Bio/Technol.8:535-542; and Finnegan et al. (1994) Bio/Technol. 12:883-888.

[0161] In the case of rice, protoplast-mediated transformation for bothJaponica- and Indica-types has been described (Zhang et al. (1988) PlantCell Rep. 7:379-384; Shimamoto et al. Nature 338:274-277; Datta et al.(1990) Biotechnol. 8:736-740) and both types are also routinelytransformable using particle bombardment. Christou et al. (1991)Biotechnol. 9:957-962.

[0162] In the case of wheat, transformation by particle bombardment hasbeen described for both type C long-term regenerable callus (Vasil etal. (1992) Biotechnol. 10:667-674) and immature embryos and immatureembryo-derived callus (Vasil et al. (1993) Biotechnol. 11:1553-1558;Weeks et al. (1993) Plant Physiol. 102:1077-1084). A further techniqueis described in published patent applications WO 94/13822 and WO95/33818.

[0163] Transformation of plant cells is normally undertaken with aselectable marker that can provide resistance to an antibiotic or to aherbicide. Selectable markers that are routinely used in transformationinclude the nptII gene which confers resistance to kanamycin (Messing &Vierra (1982) Gene 19:259-268; and Bevan et al. (1983) Nature304:184-187), the bar gene which confers resistance to the herbicidephosphinothricin (White et al. (1990) Nucl. Acids Res. 18:1062; Spenceret al. (1990) Theor. Appl. Genet. 79:625-631), the hph gene whichconfers resistance to the antibiotic hygromycin (Blochlinger andDiggelmann (1984) Mol. Cell. Biol. 4:2929-2931), and the dhfr gene whichconfers resistance to methotrexate (Bourouis et al. (1983) EMBO J.2:1099-1104). More recently, a number of selection systems have beendeveloped which do not rely of selection for resistance to antibiotic orherbicide. These include the inducible isopentyl transferase systemdescribed by Kunkel et al. (1999) Nature Biotechnol. 17:916-919.

[0164] The zinc finger protein constructs of the invention are suitablefor expression in a variety of different organisms. However, to enhancethe efficiency of expression it can be necessary to modify thenucleotide sequence encoding the zinc finger protein to account fordifferent frequencies of codon usage in different host organisms. Henceit is preferable that the sequences to be introduced into organisms,such as plants, conform to preferred usage of codons in the hostorganism.

[0165] In general, high expression in plants is best achieved from codonsequences that have a GC content of at least 35% and preferably morethan 45%. This is thought to be because the existence of ATTTA motifsdestabilize messenger RNAs and the existence of AATAAA motifs can causeinappropriate polyadenylation, resulting in truncation of transcription.Murray et al. (1989) (Nucl. Acids Res. 17:477-498) have shown that evenwithin plants, monocotyledonous and dicotyledonous species havediffering preferences for codon usage, with monocotyledonous speciesgenerally preferring GC richer sequences. Thus, in order to achieveoptimal high level expression in plants, gene sequences can be alteredto accommodate such preferences in codon usage in such a manner that thecodons encoded by the DNA are not changed.

[0166] Plants also have a preference for certain nucleotides adjacent tothe ATG encoding the initiating methionine and for most efficienttranslation, these nucleotides can be modified. To facilitatetranslation in plant cells, it is preferable to insert, immediatelyupstream of the ATG representing the initiating methionine of the geneto be expressed, a “plant translational initiation context sequence”. Avariety of sequences can be inserted at this position. These include thesequence the sequence 5′-AAGGAGATATAACAATG-3′ (SEQ ID NO: 1) (Prasher etal. (1992) Gene 111:229-233; and Chalfie et al. (1992) Science263:802-805), the sequence 5′-GTCGACCATG-3′ (SEQ ID NO: 2) (Clontech1993/1994 catalog, page 210), and the sequence 5′-TAAACAATG-3′. Joshi etal. (1987) Nucl. Acids Res. 15:6643-6653. For any particular plantspecies, a survey of natural sequences available in any databank (e.g.GenBank) can be undertaken to determine preferred “plant translationalinitiation context sequences” on a species-by-species basis.

[0167] Any changes that are made to the coding sequence can be madeusing techniques that are well known in the art and include sitedirected mutagenesis, PCR, and synthetic gene construction. Such methodsare described in published patent applications EP 0 385 962 , EP 0 359472 and WO 93/07278. Well-known protocols for transient expression inplants can be used to check the expression of modified genes beforetheir transfer to plants by transformation.

[0168] F. Regulation of Gene Expression in Vivo in Plants Using ZincFingers

[0169] The present invention provides a method of regulating geneexpression in a plant using an engineered zinc finger.

[0170] Thus, zinc fingers such as those designed or selected asdescribed above are useful in switching or modulating gene expression inplants, in particular with respect to agricultural biotechnologyapplications as described below.

[0171] A fusion polypeptide comprising a zinc finger targeting domainand a DNA cleavage domain can be used to regulate gene expressing byspecific cleavage of nucleic acid sequence. More usually, the zincfingers will be fused to a transcriptional effector domain to activateor repress transcription from a gene that possesses the zinc fingerbinding sequence in its upstream sequences. Zinc fingers capable ofdifferentiating between U and T can be used to preferentially target RNAor DNA, as required.

[0172] Thus zinc finger polypeptides according to the invention willtypically require the presence of a transcriptional effector domain,such as an activation domain or a repressor domain. Examples oftranscriptional activation domains include the VP16 and VP64transactivation domains of Herpes Simplex Virus. Alternativetransactivation domains are various and include the maize C1transactivation domain sequence (Sainz et al. (1997) Mol. Cell. Biol.17:115-22) and P1 (Goff et al. (1992) Genes Dev. 6:864-75; and Estruchet al. (1994) Nucl. Acids Res. 22:3983-89) and a number of other domainsthat have been reported from plants. Estruch et al. (1994).

[0173] Instead of incorporating a transactivator of gene expression, arepressor of gene expression can be fused to the Zinc finger protein andused to down regulate the expression of a gene contiguous orincorporating the zinc finger protein target sequence. Such repressorsare known in the art and include, for example, the KRAB-A domain(Moosmann et al. (1997) Biol. Chem. 378:669-677) the engrailed domain(Han et al. (1993) EMBO J. 12:2723-2733) and the snag domain (Grimes etal. (1996) Mol. Cell. Biol. 16:6263-6272). These can be used alone or incombination to down-regulate gene expression.

[0174] Another possible application discussed above is the use of zincfingers fused to nucleic acid cleavage moieties, such as the catalyticdomain of a restriction enzyme, to produce a restriction enzyme capableof cleaving only target DNA of a specific sequence. Kim et al. (1996)Proc. Natl. Acad. Sci. USA 93:1156-1160. Using such approaches,different zinc finger domains can be used to create restriction enzymeswith any desired recognition nucleotide sequence. Preferably, theexpression of these zinc finger-enzyme fusion proteins is inducible.Enzymes other than those that cleave nucleic acids can also be used fora variety of purposes.

[0175] The target gene can be endogenous or heterologous to the genomeof the cell, for example fused to a heterologous coding sequence.However, in either case it will comprise a target DNA sequence, such asa target DNA sequence described above, to which a zinc finger accordingto the invention binds. The zinc finger is typically expressed from aDNA construct present in the host cell comprising the target sequence.The DNA construct is preferably stably integrated into the genome of thehost cell, but this is not essential.

[0176] Thus a host plant cell according to the invention comprises atarget DNA sequence and a construct capable of directing expression ofthe zinc finger molecule in the cell.

[0177] Suitable constructs for expressing the zinc finger molecule areknown in the art and are described in section E above. The codingsequence can be expressed constitutively or be regulated. Expression canbe ubiquitous or tissue-specific. Suitable regulatory sequences areknown in the art and are also described in section E above. Thus the DNAconstruct will comprise a nucleic acid sequence encoding a zinc fingeroperably linked to a regulatory sequence capable of directing expressionof the zinc finger molecule in a host cell.

[0178] It can also be desirable to use target DNA sequences that includeoperably linked neighboring sequences that bind transcriptionalregulatory proteins, such as transactivators. Preferably thetranscriptional regulatory proteins are endogenous to the cell. If not,they will typically need to be introduced into the host cell usingsuitable nucleic acid constructs.

[0179] Techniques for introducing nucleic acid constructs into plantcells are known in the art and many are described both in section E andbelow in the section on the production of transgenic plants.

[0180] “Transgenic” in the present context denotes organisms and moreespecially plants in which one or more cells receive a recombinant DNAmolecule. Typically the transgene introduced will be transferred to thenext generation which is also thus denoted “transgenic”.

[0181] The information introduced into the organism is preferably aspecies foreign to the recipient animal (i.e., “heterologous”), but theinformation can also be foreign only to the particular individualrecipient, or genetic information already possessed by the recipient. Inthe last case, the introduced gene can be differently expressed than isthe native gene.

[0182] “Operably linked” refers to polynucleotide sequences that arenecessary to effect the expression of coding and non-coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and a transcription termination sequence. The term“control sequences” is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

[0183] Thus, a polynucleotide construct for use in the presentinvention, to introduce a nucleotide sequence encoding a zinc fingermolecule into the genome of a multicellular organism, typicallycomprises a nucleotide sequence encoding the zinc finger moleculeoperably linked to a regulatory sequence capable of directing expressionof the coding sequence. In addition the polynucleotide construct cancomprise flanking sequences homologous to the host cell organism genometo aid in integration.

[0184] G. Construction of Transgenic Plants Expressing Zinc FingerMolecules

[0185] A transgenic plant of the invention can be produced from anyplant such as the seed-bearing plants (angiosperms), and conifers.Angiosperms include dicotyledons and monocotyledons. Examples ofdicotyledonous plants include tobacco, (Nicotiana plumbaginifolia andNicotiana tabacum), arabidopsis (Arabidopsis thaliana), Brassica napus,Brassica nigra, Datura innoxia, Vicia narbonensis, Vicia faba, pea(Pisum sativum), cauliflower, carnation and lentil (Lens culinaris).Examples of monocotyledonous plants include cereals such as wheat,barley, oats and maize.

[0186] Techniques for producing transgenic plants are well known in theart. Typically, either whole plants, cells or protoplasts can betransformed with a suitable nucleic acid construct encoding a zincfinger molecule or target DNA (see above for examples of nucleic acidconstructs). There are many methods for introducing transforming DNAconstructs into cells, but not all are suitable for delivering DNA toplant cells. Suitable methods include Agrobacterium infection (see,among others, Turpen et al. (1993) J. Virol. Met. 42:227-239) or directdelivery of DNA such as, for example, by PEG-mediated transformation, byelectroporation or by acceleration of DNA coated particles. Accelerationmethods are generally preferred and include, for example,microprojectile bombardment. A typical protocol for producing transgenicplants (in particular monocotyledons), taken from U.S. Pat. No.5,874,265, is described below.

[0187] An example of a method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method,non-biological particles can be coated with nucleic acids and deliveredinto cells by a propelling force. Exemplary particles include thosecomprised of tungsten, gold, platinum, and the like.

[0188] A particular advantage of microprojectile bombardment, inaddition to it being an effective means of reproducibly stablytransforming both dicotyledons and monocotyledons, is that neither theisolation of protoplasts nor the susceptibility to Agrobacteriuminfection is required. An illustrative embodiment of a method fordelivering DNA into plant cells by acceleration is a Biolistics ParticleDelivery System, that can be used to propel particles coated with DNAthrough a screen, such as a stainless steel or Nytex screen, onto afilter surface covered with plant cells cultured in suspension. Thescreen disperses the tungsten-DNA particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat without a screen intervening between the projectile apparatus andthe cells to be bombarded, the projectiles aggregate and can be toolarge for attaining a high frequency of transformation. This can be dueto damage inflicted on the recipient cells by projectiles that are toolarge.

[0189] For the bombardment, cells in suspension are preferablyconcentrated on filters. Filters containing the cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate. If desired, one or more screens are also positionedbetween the gun and the cells to be bombarded. Through the use oftechniques set forth herein one can obtain up to 1000 or more clustersof cells transiently expressing a marker gene (“foci”) on the bombardedfilter. The number of cells in a focus which express the exogenous geneproduct 48 hours post-bombardment often range from 1 to 10 and average 2to 3.

[0190] After effecting delivery of exogenous DNA to recipient cells byany of the methods discussed above, a preferred step is to identify thetransformed cells for further culturing and plant regeneration. Thisstep can include assaying cultures directly for a screenable trait or byexposing the bombarded cultures to a selective agent or agents.

[0191] An example of a screenable marker trait is the red pigmentproduced under the control of the R-locus in maize. This pigment can bedetected by culturing cells on a solid support containing nutrient mediacapable of supporting growth at this stage, incubating the cells at,e.g., 18° C. and greater than 180 μE m⁻² s⁻¹, and selecting cells frompigmented colonies (visible aggregates of cells). These cells can becultured further, either in suspension or on solid media.

[0192] An exemplary embodiment of methods for identifying transformedcells involves exposing the bombarded cultures to a selective agent,such as a metabolic inhibitor, an antibiotic, herbicide or the like.Cells that have been transformed and have stably integrated a markergene conferring resistance to the selective agent used, will grow anddivide in culture. Sensitive cells will not be amenable to furtherculturing.

[0193] To use the bar-bialaphos selective system, bombarded cells onfilters are resuspended in nonselective liquid medium, cultured (e.g.for one to two weeks) and transferred to filters overlaying solid mediumcontaining from 1-3 mg/l bialaphos. While ranges of 1-3 mg/l willtypically be preferred, it is proposed that ranges of 0.1-50 mg/l willfind utility in the practice of the invention. The type of filter foruse in bombardment is not believed to be particularly crucial, and cancomprise any solid, porous, inert support.

[0194] Cells that survive the exposure to the selective agent can becultured in media that supports regeneration of plants. Tissue ismaintained on a basic media with hormones for about 2-4 weeks, thentransferred to media with no hormones. After 2-4 weeks, shootdevelopment will signal the time to transfer to another media.

[0195] Regeneration typically requires a progression of media whosecomposition has been modified to provide the appropriate nutrients andhormonal signals during sequential developmental stages from thetransformed callus to the more mature plant. Developing plantlets aretransferred to soil, and hardened, e.g., in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and 250μE m⁻² s−1 of light. Plants are preferably matured either in a growthchamber or greenhouse. Regeneration will typically take about 3-12weeks. During regeneration, cells are grown on solid media in tissueculture vessels. An illustrative embodiment of such a vessel is a petridish. Regenerating plants are preferably grown at about 19° C. to 28° C.After the regenerating plants have reached the stage of shoot and rootdevelopment, they can be transferred to a greenhouse for further growthand testing.

[0196] Genomic DNA can be isolated from callus cell lines and plants todetermine the presence of the exogenous gene through the use oftechniques well known to those skilled in the art such as PCR and/orSouthern blotting.

[0197] Several techniques exist for inserting the genetic information,the two main principles being direct introduction of the geneticinformation and introduction of the genetic information by use of avector system. A review of the general techniques can be found inarticles by Potrykus ((1991) Annu. Rev. Plant Physiol. Plant Mol. Biol.42:205-225) and Christou (Agri-Food-Industry Hi-Tech Mar./Apr. 17-27,1994).

[0198] Thus, in one aspect, the present invention relates to a vectorsystem that carries a construct encoding a zinc finger molecule ortarget DNA according to the present invention and that is capable ofintroducing the construct into the genome of a plant.

[0199] The vector system can comprise one vector, but it can comprise atleast two vectors. In the case of two vectors, the vector system isnormally referred to as a binary vector system. Binary vector systemsare described in further detail in Gynheung An et al. (1980), BinaryVectors, Plant Molecular Biology Manual A3, 1-19.

[0200] One extensively employed system for transformation of plant cellswith a given promoter or nucleotide sequence or construct is based onthe use of a Ti plasmid from A. tumefaciens or a Ri plasmid fromAgrobacterium rhizogenes (An et al. (1986) Plant Physiol. 81, 301-305and Butcher et al. (1980) Tissue Culture Methods for Plant Pathologists,eds.:D. S. Ingrams and J. P. Helgeson, 203-208).

[0201] Several different Ti and Ri plasmids have been constructed thatare suitable for the construction of the plant or plant cell constructsdescribed above.

[0202] H. Examples of Specific Applications

[0203] Zinc fingers according to the invention can be used to regulatethe expression of a nucleotide sequence of interest in the cell of aplant. Applications of the present invention include the overexpressionor turning off of any desired target gene (which could be a gene orgene/s in a biosynthetic pathway or a transcription factor), or theinvestigation of the function of a gene in the plant. Some specificapplications include the following:

[0204] 1. Improvement of traits that are of interest to processors andend users of plants. For example, modifying the expression of genesinvolved in starch or oil biosynthsis (for example) will provide plantswith improved processing and end-user characteristics.

[0205] 2. Improvement in the plant use of inorganic nutrients. Bymodifying the expression of key enzymes involved in nutrient use, cropplants may be better able to grow in environments of lower nutrientavailability or with the application of less inorganic fertilizer.

[0206] 3. Improvement of other characteristics by manipulation of plantgene expression. Overexpression of the Na+/H+ antiport gene has resultedin enhanced salt tolerance in Arabidopsis. Targeted zinc fingers can beused to regulate the endogenous gene.

[0207] 4. Improvement of ripening characteristics in fruit. A number ofgenes have been identified that are involved in the ripening process(such as in ethylene biosynthesis). Control of the ripening process viaregulation of the expression of those genes will help reduce significantlosses via spoilage.

[0208] 5. Modification of plant growth characteristics throughintervention in hormonal pathways. Many plant characteristics arecontrolled by hormones. Regulation of the genes involved in theproduction of and response to hormones will enable produce crops withaltered characteristics.

[0209] 6. Improvement of plant aroma and flavor. Pathways leading to theproduction of aroma and flavor compounds in vegetables and fruit arecurrently being elucidated allowing the enhancement of these traitsusing zinc finger technology.

[0210] 7. Improving the pharmaceutical and nutraceutical potential ofplants. Many pharmaceutically active compounds are known to exist inplants, but in many cases production is limited due to insufficientbiosynthesis in plants. Zinc finger technology could be used to overcomethis limitation by upregulating specific genes or biochemical pathways.Other uses include regulating the expression of genes involved inbiosynthesis of commercially valuable compounds that are toxic to thedevelopment of the plant.

[0211] 8. Reducing harmful plant components. Some plant components leadto adverse allergic reaction when ingested in food. Zinc fingertechnology could be used to overcome this problem by downregulatingspecific genes responsible for these reactions.

[0212] 9. As well as modulating the expression of endogenous genes,heterologous genes can be introduced whose expression is regulated byzinc finger proteins. For example, a nucleotide sequence of interest canencode a gene product that is preferentially toxic to cells of the maleor female organs of the plant such that the ability of the plant toreproduce can be regulated. Alternatively, or in addition, theregulatory sequences to which the nucleotide sequence is operably linkedcan be tissue-specific such that expression when induced only occurs inmale or female organs of the plant. Suitable sequences and/or geneproducts are described in WO89/10396, WO92/04454 (the TA29 promoter fromtobacco) and EP-A-344,029, EP-A-412,006 and EP-A-412,911.

[0213] The present invention will now be described by way of thefollowing examples, which are illustrative only and non-limiting. TheExamples show that a zinc finger chimera can be expressed in plants andrecognize a determined sequence in a plant genome. Secondly, chimerascontaining a transactivating domain can activate the expression of areported gene in plants in a manner similar to animal cells. Using thisprinciple, zinc fingers can be designed to interact with specificsequences in plant genomes to either activate or repress the expressionof genes of interest.

EXAMPLES Materials and Methods Gene Construction and Cloning

[0214] In general, procedures and materials are in accordance withguidance given in Sambrook et al. Molecular Cloning. A LaboratoryManual, Cold Spring Harbor, 1989. The gene for the Zif268 fingers(residues 333-420) is assembled from 8 overlapping syntheticoligonucleotides (see Choo and Klug (1994)), giving SfiI and NotIoverhangs. The genes for fingers of the phage library are synthesizedfrom 4 oligonucleotides by directional end to end ligation using 3 shortcomplementary linkers, and amplified by PCR from the single strand usingforward and backward primers which contain sites for NotI and SfiIrespectively. Backward PCR primers in addition introduce Met-Ala-Glu asthe first three amino acids of the zinc finger peptides, and these arefollowed by the residues of the wild type or library fingers asrequired. Cloning overhangs are produced by digestion with SfiI and NotIwhere necessary. Fragments are ligated to 1 μg similarly preparedFd-Tet-SN vector. This is a derivative of fd-tet-DOG1 (Hoogenboom et al.(1991) Nucl. Acids Res. 19:4133-4137) in which a section of the pelBleader and a restriction site for the enzyme SfiI (underlined) have beenadded by site-directed mutagenesis using the oligonucleotide:

[0215] 5′ CTCCTGCAGTTGGACCTGTGCCATGGCCGGCTGGGCCGCATAGAATGG AACAACTAAAGC3′ (SEQ ID NO: 3)

[0216] that anneals in the region of the polylinker. ElectrocompetentDH5α cells are transformed with recombinant vector in 200 ng aliquots,grown for 1 hour in 2×TY medium with 1% glucose, and plated on TYEcontaining 15 μg/ml tetracycline and 1% glucose.

[0217] The zinc finger chimera that we used for this first set ofexperiments is a fusion protein that comprises 4 domain. First, 4fingers of TFIIIA were linked through a spacer region to 3 fingers ofZif268 and this is denoted TFIIIAZif. Choo and Klug (1997) Curr. Opin.Str. Biol. 7:117-125; Pavletich and Pabo (1991) Science 252:809-817;Elrod-Erickson et al. (1996) Structure 4:1171-1180; and Elrod-Ericksonet al (1998) Structure 6:451-464. This designed zinc finger is able torecognize specifically a DNA sequence of 27 base pair (bp). Second, ashort region (NLS) that is a nuclear localization region rich in basicamino acids that directs the chimera to the nucleus. Third, atransactivation domain from the Herpes Simplex Virus (HSV) VP16 or VP64that is a tetramer of the minimal VP16 domain. This region activatesgene expression. Last, the 9E10 region that correspond to the myc domainfor the specific antibody recognition of the expressed protein in plants(FIG. 3).

[0218] The reporter construct consists of a DNA monomer of the minimalbinding site of 27 bp or tetramer of the minimal region that isrecognized specifically by the zinc finger domain. This sequence isattached 5′ to the 46 bp of the CaMV 35S minimal. Downstream of thepromoter we have cloned the coding sequence luciferase or greenfluorescent protein (GFP) genes as reporter genes. The luciferase fromPhotinus pyralis catalyzes the ATP/oxygen-dependent oxidization ofsubstrate luciferin which produces the emission of light(bioluminescence) and the GFP fluoresces under blue light. At 3′ end ofthe construct contains the pea rbcS-E9 polyadenylation sequence (FIG.3).

[0219] The zinc finger phage display library of the present inventioncontains amino acid randomizations in putative base-contacting positionsfrom the second and third zinc fingers of the three-finger DNA bindingdomain of Zif268, and contains members that bind DNA of the sequenceXXXXXGGCG where X is any base. Further details of the library used canbe found in WO 98/53057.

Example 1 Generation of Transgenic Plants Expressing a Zinc FingerProtein Fused to a Transactivation Domain

[0220] To investigate the utility of heterologous zinc finger proteinsfor the regulation of plant genes, a synthetic zinc finger protein wasdesigned and introduced into transgenic A. thaliana under the control ofa promoter capable of expression in a plant as described below. A secondconstruct comprising the zinc finger protein binding sequence fusedupstream of the Green Fluorescent Protein (GFP) reporter gene was alsointroduced into transgenic A. thaliana as described in Example 2.Crossing the two transgenic lines produced progeny plants carrying bothconstructs in which the GFP reporter gene was expressed demonstratingtransactivation of the gene by the zinc finger protein.

[0221] Using conventional cloning techniques, the following constructswere made as XbaI-BamHI fragments in the cloning vector pcDNA3.1(Invitrogen).

pTFIIIAZifVP16

[0222] pTFIIIAZifVP16 comprises a fusion of four finger domains of thezinc finger protein TFIIIA fused to the three fingers of the zinc fingerprotein Zif268. The TFIIIA-derived sequence is fused in frame to thetranslational initiation sequence ATG. The 7 amino acid NuclearLocalization Sequence (NLS) of the wild-type Simian Virus 40 LargeT-Antigen is fused to the 3′ end of the Zif268 sequence, and the VP16transactivation sequence is fused downstream of the NLS. In addition, 30bp sequence from the c-myc gene is introduced downstream of the VP16domain as a “tag” to facilitate cellular localization studies of thetransgene. While this is experimentally useful, the presence of this tagis not required for the activation (or repression) of gene expressionvia zinc finger proteins.

[0223] The sequence of pTFIIIAZifVP16 is shown in SEQ ID NO: 4 as anXbaI-BamHI fragment. The translational initiating ATG is located atposition 15 and is double underlined. Fingers 1 to 4 of TFIIIA extendfrom position 18 to position 416. Finger 4 (positions 308-416) does notbind DNA within the target sequence, but instead serves to separate thefirst three fingers of TFIIIA from Zif268 which is located at positions417-689. The NLS is located at positions 701-722, the VP16transactivation domain from positions 723-956, and the c-myc tag frompositions 957-986. This is followed by the translational terminator TAA.

pTFIIIAZifVP64

[0224] pTFIIIAZifVP64 is similar to pTFIIIAZifVP16 except that the VP64transactivation sequence replaces the VP16 sequence of pTFIIIAZifVP16.

[0225] The sequence of pTFIIIAZifVP64 is shown in SEQ ID NO: 5 as anXbaI-BamHI fragment. Locations within this sequence are as forpTFIIIAZifVP16 except that the VP64 domain is located at position723-908 and the c-myc tag from positions 909-938.

[0226] The DNA binding site for the TFIIIAZif protein contains the DNArecognition sites for zinc fingers 1-3 of TFIIIA and the three zincfingers of Zif 268. These are the DNA sequences GGATGGGAGAC andGCGTGGGCGT, respectively. The six base pair sequence GTACCT in SequenceID NO: 3 is a spacer region of DNA that separates the two binding sitesand the nucleotide composition of the DNA spacer appears to have noeffect on binding of the protein. Therefore, this or other structuredlinkers could be used with other DNA spacers of different length andsequence.

[0227] The amino acid sequence of zinc Finger 4 of TFIIIA, including theflanking sequences as used in the composite protein of the invention, is

[0228] NIKICVYVCHFENCGKAFKKHNQLK VHQFSHTQQLP.

[0229] The nucleotide Sequence of Zinc Finger 4 of TFIIIA, including theflanking sequences, isAACATCAAGATCTGCGTCTATGTGTGCCATTTTGAGAACTGTGGCAAAGCATTCAAGAAACACAATCAATTAAAGGTTCATCAGTTCAGTCACACACAGCAGCTGCCG.

[0230] Using conventional cloning techniques, the sequence5′-AAGGAGATATAACA-3′ (SEQ ID NO: 6) is introduced upstream of thetranslational initiating ATG of both pTFIIIAZifVP16 and pTFIIIAZifVP64.This sequence incorporates a plant translational initiation contextsequence to facilitate translation in plant cells. Prasher et al. Gene111:229-233 (1992); and Chalfie et al. Science 263:802-805 (1992).

[0231] The final constructs are transferred to the plant binary vectorpBIN121 between the Cauliflower Mosaic Virus 35S promoter and thenopaline synthase terminator sequence. This transfer is effected usingthe XbaI site of pBIN121. The binary constructs thus derived are thenintroduced into A. tumefaciens (strain LBA 4044 or GV 3101) either bytriparental mating or direct transformation.

[0232] Next, A. thaliana are transformed with Agrobacterium containingthe binary vector construct using conventional transformationtechniques. For example, using vacuum infiltration (e.g. Bechtold et al.CR Acad. Sci. Paris 316:1194-1199; Bent et al. (1994) Science265:1856-1860), transformation can be undertaken essentially as follows.Seeds of Arabidopsis are planted on top of cheesecloth covered soil andallowed to grow at a final density of 1 per square inch under conditionsof 16 hours light/8 hours dark. After 4-6 weeks, plants are ready toinfiltrate. An overnight liquid culture of Agrobacterium carrying theappropriate construct is grown up at 28° C. and used to inoculate afresh 500 ml culture. This culture is grown to an OD₆₀₀ of at least 2.0,after which the cells are harvested by centrifugation and resuspended in1 liter of infiltration medium (1 liter prepared to contain:2.2 g MSSalts, 1×B5 vitamins, 50 g sucrose, 0.5 g MES pH 5.7, 0.044 μMbenzylaminopurine, 200 L Silwet μL-77 (OSI Specialty)). To vacuuminfiltrate, pots are inverted into the infiltration medium and placedinto a vacuum oven at room temperature. Infiltration is allowed toproceed for 5 mins at 400 mm Hg. After releasing the vacuum, the pot isremoved and laid it on its side and covered with Saran™ wrap. The coveris removed the next day and the plant stood upright. Seeds harvestedfrom infiltrated plants are surface sterilized and selected onappropriate medium. Vernalization is undertaken for two nights at around4° C. Plates are then transferred to a plant growth chamber. After about7 days, transformants are visible and are transferred to soil and grownto maturity.

[0233] Transgenic plants are grown to maturity. They appearphenotypically normal and are selfed to homozygosity using standardtechniques involving crossing and germination of progeny on appropriateconcentration of antibiotic.

[0234] Transgenic plant lines carrying the TFIIIAZifVP16 construct aredesignated At-TFIIIAZifVP16 and transgenic plant lines carrying theTFIIIAZifVP64 construct are designated At-TFIIIAZifVP64.

Example 2 Generation of Transgenic Plants Carrying a Green FluorescentProtein Reporter Gene

[0235] A reporter plasmid is constructed which incorporates the targetDNA sequence of the TFIIIAZifVP16 and TFIIIAZifVP64 zinc finger proteinsdescribed above upstream of the Green Fluorescent Protein (GFP) reportergene. The target DNA sequence of TFIIIAZifVP16 and TFIIIAZifVP64 isshown in SEQ ID NO: 7.

[0236] This sequence is incorporated in single copy immediately upstreamof the CaMV 35S −90 or −46 minimal promoter to which the GFP gene isfused.

[0237] The resultant plasmid, designated pTFIIIAZif-UAS/GFP, istransferred to the plant binary vector pBIN121 replacing the CauliflowerMosaic Virus 35S promoter. This construct is then transferred to A.tumefaciens and subsequently transferred to A. thaliana as describedabove. Transgenic plants carrying the construct are designatedAt-TFIIIAZif-UAS/GFP.

Example 3 Use of Zinc Finger Proteins to Up-Regulate a Transgene in aPlant

[0238] To assess whether the zinc finger constructs TFIIIAZifVP16 andTFIIIAZifVP64 are able to transactivate gene expression in planta,Arabidopsis lines At-TFIIIAZifVP16 and At-TFIIIAZifVP64 are crossed toAt-TFIIIAZif-UAS/GFP. The progeny of such crosses yield plants thatcarry the reporter construct TFIIIAZif-UAS/GFP together with either thezinc finger protein construct TFIIIAZifVP16 or the zinc finger constructTFIIIAZifVP64.

[0239] Plants are screened for GFP expression using an invertedfluorescence microscope (Leitz DM-IL) fitted with a filter set (Leitz-Dexcitation BP 355-425, dichronic 455, emission LP 460) suitable for themain 395 nm excitation and 509 nm emission peaks of GFP.

[0240] In each case, the zinc finger construct is able to transactivategene expression demonstrating the utility of heterologous zinc fingerproteins for the regulation of plant genes.

Example 4 Generation of Transgenic Plants Expressing a Zinc Finger Fusedto a Plant Transactivation Domain

[0241] The constructs pTFIIIAZifVP16 and pTFIIIAZifVP64 utilize the VP16and VP64 transactivation domains of Herpes Simplex Virus to activategene expression. Alternative transactivation domains are various andinclude the C1 transactivation domain sequence (from maize; see Goff etal. (1991) Genes Dev. 5:298-309; Goff et al. (1992) Genes Dev.6:864-875), and a number of other domains that have been reported fromplants. Estruch et al. (1994) Nucl. Acids Res. 22:3983-3989.

[0242] Construct pTFIIAZifC1 is made as described above forpTFIIIAZifVP16 and pTFIIIAZifVP64 except the VP16/VP64 activationdomains are replaced with the C1 transactivation domain sequence

[0243] A transgenic Arabidopsis line, designated At-TFIIAZifC1, isproduced as described above in Example 2 and crossed withAt-TFIIIAZif-UAS/GFP. The progeny of such crosses yield plants thatcarry the reporter construct TFIIIAZif-UAS/GFP together with either thezinc finger protein construct TFIIIAZifC1.

[0244] Plants are screened for GFP expression using an invertedfluorescence microscope (Leitz DM-IL) fitted with a filter set (Leitz-Dexcitation BP 355-425, dichronic 455, emission LP 460) suitable for themain 395 nm excitation and 509 nm emission peaks of GFP.

Example 5 Regulation of an Endogenous Plant Gene—UDP Glucose FlavanoidGlucosyl-Transferase (UFGT).

[0245] To determine whether a suitably configured zinc finger could beused to regulate gene transcription from an endogenous gene in a plant,the maize UDP glucose flavanoid glucosyl-transferase (UFGT) gene (theBronzel gene) was selected as the target gene. UFGT is involved inanthocyanin biosynthesis. A number of wild type alleles have beenidentified including Bz-W22 that conditions a purple phenotype in themaize seed and plant. The Bronze locus has been the subject of extensivegenetic research because its phenotype is easy to score and itsexpression is tissue-specific and varied (for example aleurone, anthers,husks, cob and roots). The complete sequence of Bz-W22 includingupstream regulatory sequences has been determined (Ralston et al.Genetics 119:185-197). A number of sequence motifs that bindtranscriptional regulatory proteins have been identified within theBronze promoter including sequences homologous to consensus bindingsites for the myb- and myc-like proteins (Roth et al. Plant Cell3:317-325). Identification of a zinc finger that binds to the bronzepromoter

[0246] The first step is to carry out a screen for zinc finger proteinsthat bind to a selected region of the Bronze promoter. A region ischosen just upstream of the AT rich block located at between −88 and−80, which has been shown to be critical for Bz1 expression (Roth et al.supra).

[0247] 1. Bacterial colonies containing phage libraries that express alibrary of Zif268 zinc fingers randomized at one or more DNA bindingresidues are transferred from plates to culture medium. Bacterialcultures are grown overnight at 30° C. Culture supernatant containingphages is obtained by centrifugation.

[0248] 2. 10 pmol of biotinylated target DNA, derived from the Bronzepromoter, immobilized on 50 mg streptavidin beads (Dynal) is incubatedwith I ml of the bacterial culture supernatant diluted 1:1 with PBScontaining 50 μM ZnCl₂, 4% Marvel, 2% Tween in a streptavidin coatedtube for 1 hour at 20° C. on a rolling platform in the presence of 4 μgpoly (d(I-C)) as competitor.

[0249] 3. The tubes are washed 20 times with PBS containing 50 μM ZnCl2and 1% Tween, and 3 times with PBS containing 50 μM ZnCl₂ to removenon-binding phage.

[0250] 4. The remaining phage are eluted using 0.1 ml 0.1 Mtriethylamine and the solution is neutralized with an equal volume of 1M Tris-Cl (pH 7.4).

[0251] 5. Logarithmic-phase E. coli TG1 cells are infected with elutedphage, and grown overnight, as described above, to prepare phagesupernatants for subsequent rounds of selection.

[0252] 6. Single colonies of transformants obtained after four rounds ofselection (steps 1 to 5) as described, are grown overnight in culture.Single-stranded DNA is prepared from phage in the culture supernatantand sequenced using the Sequenase™ 2.0 kit (U.S. Biochemical Corp.). Theamino acid sequences of the zinc finger clones are deduced.

[0253] Construction of a vector for expression of the zinc finger clonefused to a C1 activation domain in maize protoplasts

[0254] Using conventional cloning techniques and in a similar manner toExample 1, the construct pZifBz23C1 is made in cloning vector pcDNA3.1(Invitrogen).

[0255] pZifBz23C1 comprises the three fingers of the zinc finger proteinclone ZifBz23 fused in frame to the translational initiation sequenceATG. The 7 amino acid Nuclear Localization Sequence (NLS) of thewild-type Simian Virus 40 Large T-Antigen is fused to the 3′ end of theZifBz23 sequence, and the C1 transactivation sequence is fuseddownstream of the NLS. In addition, 30 bp sequence from the c-myc geneis introduced downstream of the VP16 domain as a “tag” to facilitatecellular localization studies of the transgene.

[0256] The coding sequences of pZifBz23C1 are transferred to a plantexpression vector suitable for use in maize protoplasts, the codingsequence being under the control of a constitutive CaMV 35S promoter.The resulting plasmid is termed pTMBz23. The vector also contains ahygromycin resistance gene for selection purposes.

[0257] A suspension culture of maize cells is prepared from calliderived from embryos obtained from inbred W22 maize stocks grown toflowering in a greenhouse and self pollinated using essentially theprotocol described in EP-A-332104 (Examples 40 and 41). The suspensionculture is then used to prepare protoplasts using essentially theprotocol described in EP-A-332104 (Example 42).

[0258] Protoplasts are resuspended in 0.2 M mannitol, 0.1% w/v MES, 72mM NaCl, 70 mM CaCl₂, 2.5 mM KCl, 2.5 mM glucose pH to 5.8 with KOH, ata density of about 2×10⁶ per ml. 1 ml of the protoplast suspension isthen aliquotted into plastic electroporation cuvettes and 10 μg oflinearized pTMBz23 added. Electroporation is carried out s described inEP-A-332104 (Example 57). Protoplasts are cultured followingtransformation at a density of 2×10⁶ per ml in KM-8p medium with nosolidifying agent added.

[0259] Measurements of the levels UFGT expression are made usingcolorimetry and/or biochemical detection methods such as Northern blotsor the enzyme activity assays described by Dooner and Nelson (1977)Proc. Natl. Acad. Sci. USA 74:5623-5627. Comparison is made with mocktreated protoplasts transformed with a vector only control.

[0260] Alternatively, or in addition to, analyzing expression of UFGT intransformed protoplasts, intact maize plants can be recovered fromtransformed protoplasts and the extent of UFGT expression determined.Suitable protocols for growing up maize plants from transformedprotoplasts are known in the art. Electroporated protoplasts areresuspended in Km-8p medium containing 1.2% w/v Seaplaque agarose and 1mg/l 2,4-D. Once the gel has set, protoplasts in agarose are place inthe dark at 26° C. After 14 days, colonies arise from the protoplasts.The agarose containing the colonies is transferred to the surface of a 9cm diameter petri dish containing 30 ml of N6 medium (EP-A-332,104)containing 2,4-D solidified with 0.24% Gelrite®. 100 mg/l hygromycin Bis also added to select for transformed cells. The callus is culturedfurther in the dark at 26° C. and callus pieces subcultured every twoweeks onto fresh solid medium. Pieces of callus can be analyzed for thepresence of the pTMBz23 construct and/or UFGT expression determined.

[0261] Corn plants are regenerated as described in Example 47 ofEP-A-332,104. Plantlets appear in 4 to 8 weeks. When 2 cm tall,plantlets are transferred to ON6 medium (EP-A-332,104) in GA7 containersand roots form in 2 to 4 weeks. After transfer to peat pots plants soonbecome established and can then be treated as normal corn plants.

[0262] Plantlets and plants can be assayed for UFGT expression asdescribed above.

Example 6 Cloning of Zinc Finger Chimera in the Expression Vectors

[0263] In the following Examples, zinc finger chimeras speciallydesigned for binding to specific DNA sequences in plants, wereengineered with an effector domain (transactivator or repressor) andexpressed in plants using a series of inducible gene expression systemXVE or ZVE1. Aoyama and Chua (1997); and Zou and Chua (2000). Planttransformation was performed using standard procedures utilizingAgrobacterium.

[0264] The general strategy is outlined in FIG. 1 and more specificallyin FIG. 2.

[0265] The zinc finger chimeras were expressed either under the controlof an estrogen receptor-based chemical-inducible system (binary vectorpER8, Zuo et al. (2000) Plant J. 24:265-273) or the constitutive CaMV35Spromoter (binary vector pBa002, Hajdukiewicz et al. (1994) Plant Mol.Biol. 25:989-994). The pBa002 plasmid was digested with MluI and SpeI(New England BioLabs, Mass.). The coding region of Zinc finger chimeragenes (VP16 and VP64) were engineered by PCR to have a MluI restrictionsite at the 5′ end and a SpeI site at the 3′ end. The sequences of theforward and the reverse primers were

[0266] 5′ CCACGCGTCCATGGGAGAGAAGGCGCTGCCGGTGG 3′ (SEQ ID NO: 8) and

[0267] 5′ CCACTAGTCCTTACAGATCTTCTTCAGAAATAAGTTTTTGTTCC 3′ (SEQ ID NO:9), respectively. The PCR-amplified DNA fragment was digested with MluIand SpeI, gel purified using the Qiaquick Gel extraction protocol(Qiagen, Valencia, Calif.), and ligated into the pBa002 plasmid using T4DNA ligase (New England Biolabs, Mass.). A clone for each construct wasverified by restriction analysis.

[0268] All constructs were introduced into A. tumefaciens strain ABI.Aoyama and Chua (1997). Similar procedures were used to clone the zincfinger chimeras (VP16 and VP64) into the AscI and SpeI sites. The codingregion of each Zinc finger chimera gene (VP16 and VP64) was engineeredby PCR to have an AscI restriction site at the 5′ end and a SpeI site atthe 3′ end. Reporter construct

[0269] pKL+1 plasmid was used for the construction of reporter plasmids.Foster and Chua (1999) Plant J. 17:363-372. pKL+1 plasmid contains aminimal promoter region from CaMV 35S promoter (−46 nucleotides)upstream of the luciferase coding sequence, that is terminated by pearbcS-E9 polyadenylation sequence. The pKL+1 plasmid was digested withXbaI and HindIII (New England BioLabs, Mass.). A tetramer of the DNAbinding site of the Zinc finger chimera was engineered by annealing twocomplementary oligos. The oligos were designed to have an XbaIrestriction site at the 5′ end and a HindIII site at the 3′ end. Thesequence of the sense and anti-sense strand primers were

[0270] 5′ CCTCTAGATCGGTCTCCCATCCAGGTACACGCCCACGCAAGTCGGTCTCCCATCCAGGTACACGCCCACGCAAGTCGGTCTCCCATCCAGGTACACGCCCACGCAAGTCGGTCTCCCATCCAGGTACACGCCCACGCAAGAAGCTTCC 3′ (SEQ ID NO: 10)

[0271] and 5′GGAAGCTTCTTGCGTGGGCGTGTACCTGGATGGGAGACCGACTTGCGTGGGCGTGTACCTGGATGGGAGACCGACTTGCGTGGGCGTGTACCTGGATGGGAGACCGACTTGCGTGGGCGTGTACCTGGATGGGAGACCGATCTAGAGG3′ (SEQ ID NO: 11),respectively. The oligos were heated to 100° C. temperature for 5 min inTE (10 mM Tris-HCl pH8.5, EDTA 1 mM) solution containing 500 mM NaCl andcooled to room temperature. The annealed oligos were isolated from anagarose gel using the Qiaquick Gel extraction protocol (Qiagen,Valencia, Calif.). The double stranded DNA fragment was digested withXbaI and HindIII, gel purified using the Qiaquick Gel extractionprotocol (Qiagen, Valencia, Calif.), and ligated into the pKL+1 plasmidusing T4 DNA ligase (New England Biolabs, Mass.). A clone for eachconstruct was verified by restriction analysis. Similar procedures wereused to engineer a single binding site reporter construct except thatthe oligos used contained an XbaI restriction site at the 5′ end and aHindIII site at the 3′ end. The sequences of the forward and the reverseprimers were 5° CCAGATCTGGTCTCCCATCCAGGTACACGCCCACGCAAGATCTCC3′ (SEQ IDNO: 12) and

[0272] 5′ GGAGATCTTGCGTGGGCGTGTACCTGGATGGGAGACCAGATCTCGG3′ (SEQ ID NO:13), respectively.

[0273] For the versions of pKL+1 plasmid containing the GFP (greenfluorescent protein) and RFP (red fluorescent protein), pKL+1 plasmidwas digested with NcoI and KpnI for GFP and SalI and KpnI for RFP (NewEngland BioLabs, Mass.). The coding region of GFP was engineered by PCRto have an NcoI restriction site at the 5′ end and an EcoRI site at the3′ end. The sequences of the forward and the reverse primers were

[0274] 5′ CCCCATGGTGAGCAAGGGCGAGGAGCTGTTCACC, 3′ (SEQ ID NO: 14) and

[0275] 5′ CCGAATTCTTACTTGTACAGCTCGTCCATGCCGAG 3′ (SEQ ID NO: 15),respectively. The coding region of RFP was engineered by PCR to have aSalI restriction site at the 5′ end and an EcoRI site at the 3′ end. Thesequences of the forward and the reverse primers were

[0276] 5′ CCCTCGAGCGGGGTACCGCGGGCCCGGG3′ (SEQ ID NO: 16) and

[0277] 5′ CAGTTGGAATTCTAGAGTCGCGGCCGCTAC3′ (SEQ ID NO: 17),respectively.

Example 7 Construction of the pZVE Plasmids

[0278] The new binary transformation plasmid pER12 (Zuo et al. 2000) wasmodified by replacing the LexA DNA binding domain with the Zinc fingerDNA binding domain (TFIIIAZif, FIG. 3). The coding region of theVP16-estrogen receptor was engineered by PCR to have XhoI restrictionsites at both the 5′ and 3′ end. The sequences of the forward and thereverse primers were 5′ CCGCTCGAGGCCCCCCCGACCGATGTCAGCCTGGGGGA3′ (SEQ IDNO: 18) and 5′ CCG CTCGAGTATTAATTTGAGAATGAACAAAAAGGACC3′ (SEQ ID NO:19), respectively. The PCR-amplified DNA fragment was digested withXhoI, gel purified using the Qiaquick Gel extraction protocol (Qiagen,Valencia, Calif.), and ligated into the pTFIIIAZif plasmid (previouslydigested with XhoI) using T4 DNA ligase (New England Biolabs, Mass.). Aclone was verified by restriction analysis and sequencing (pTFIIIAZif-VP16-ER). The coding region of the TFIIIAZif-VP16-estrogen receptorfusion gene was engineered by PCR to have an AseI restriction site atboth the 5′ and 3′ end. The sequence of the forward and the reverseprimers were 5′ GCCATTAATCGGAATGGGAGAGAAGGCGCTGCCGGTGG3′ (SEQ ID NO: 20)and 5′GCCTATTAATTTGAGAATGAACAAAAAGGACC3′ (SEQ ID NO: 21), respectively.pER12 plasmid was digested with AseI to removed the LexA-VP16-ER region.The PCR-amplified DNA fragment was digested with AseI, gel purifiedusing the Qiaquick Gel extraction protocol (Qiagen, Valencia, Calif.),and ligated into the AseI-digested pER12 plasmid using T4 DNA ligase(New England Biolabs, Mass.). A clone was verified by restrictionanalysis and sequencing. The resulting plasmid (PER8 TFIIIAZif, see FIG.3) containing Zinc finger fusion protein was digested with SalI toremove the hexamer of the LexA binding site.

[0279] The DNA fragment containing the plasmid was gel purified usingthe Qiaquick Gel extraction protocol (Qiagen, Valencia, Calif.), andligated to a double strand tetramer of zinc finger DNA binding sites(previously digested with SalI) using T4 DNA ligase (New EnglandBiolabs, Mass.). A clone was verified by restriction analysis andsequencing. The coding region of the GFP was engineered by PCR to havean XhoI restriction site at both the 5′ and 3′ end and cloned in themultiple cloning site of the vector (FIG. 4).

Example 8 Plant Transformation

[0280]Arabidopsis thaliana ecotype Landsberg erecta were transformedwith Agrobacterium using the vacuum infiltration procedure according toBent et al. (1 994) Science 265:1856-1860. Seeds collected from thevacuum infiltrated plants were surface-sterilized by treatment with asolution of 1.5% sodium hypochlorite/0.01% Tween-20 (Sigma, St LouisMo., USA) for 10 min and washing three times with sterile water. Thesterilized seeds were then resuspended in 0.1% agarose and sown in Petridishes containing A medium (full-strength Murashige and Skoog salts, pH5.7, 1% sucrose, solidified with 0.8% Bactoagar, Gibco BRL, GrandIsland, N.Y.) and 20 μg/ml hygromycin B (Sigma, St. Louis, Mo.). Theplated seeds were vernalized for 4 d and then transferred to a growthchamber maintained at 22° C. under long day conditions (16 h light/8 hdark). Transgenic T1 seedlings were selected on a plate containinghygromycin (20 mg/ml) and after 2-3 weeks of growth the presence of thetransgene was confirmed by PCR analysis. The results are presented inTable 1. TABLE 1 Constructs Onion/Trans Trans/Plants 35sVP16/4xBSLUCPositive — 35sVP16/1xBSLUC Positive — 35sVP64/4xBSLUC Positive —35sVP64/1xBSLUC Positive — ERVP16/4xBSLUC Positive(+) T1 gene(few)ERVP64/4xBSLUC Positive(+) T1 gene(few) 4xBSLUC — T1 gene(good)

Example 9 Estrogen Treatments

[0281] β-17-Estradiol (Sigma, St Louis Mo.) was dissolved indimethylsulfoxide (DMSO) to make a 100 mM stock solution. The solutionwas stored at −20° C. To monitor transgene expression, transgenic seedswere surface sterilized and sown in Petri dishes as described above.After vernalization at 4° C. for 4 days, the plates were incubated fortwo weeks in a growth chamber maintained at 22° C. under long day (16 hrlight/8 hr dark) conditions. Seedlings were removed from the plates andgrown for 2 days in a hydroponic system containing liquid A media(full-strength Murashige and Skoog salts, pH 5.7, 1% sucrose, Gibco BRL,N.Y., Dr.Takashi Aoyama personal communication). Fresh medium containingeither β-17-Estradiol (30 μM) was added and plants were removed at thedesignated time points, and then washed and frozen in liquid nitrogen.For the vector control transgenic lines similar conditions were used andthe experiments were performed in parallel. RNA analysis

[0282] Total RNA was isolated from seedlings and adult plants using theQiagen RNA purification kit (Qiagen, Valencia, Calif.). RNA gel blotanalysis was carried out according to the method described by Ausubel(1994). Each lane contained 10 μg of total RNA. The zinc finger gene,luciferase and 18S rDNA fragments were obtained by PCR amplificationwith Pfu polymerase as described above. Fragments were purified usingthe Qiaquick Gel extraction protocol (Qiagen, Valencia, Calif.). All DNAfragments were labeled with ³²P-dCTP and ³²P-dATP by random priming(Amersham, Arlington Heights, Ill.). Hybridization signals werequantified using the Phosphoimager STORM system (Molecular Dynamics) andthe data analyzed with the Image Quant v1.1 program.

Light Microscope and Luciferase Imaging

[0283] The GFP and RFP fluorescent microscopy analysis was done using anAxioskop (Zeiss, Germany) according to methods described by Mayer et al.(1993). Onion peels and 3 week old seedlings were sprayed with 2.5 mMluciferin (Promega) containing 0.005% Triton X-100 and the luciferaseactivity monitored by photon counting. Video images (5min) were capturedin gravity mode using a intensifying CCD camera and coupling MethaMorphsoftware (Universal Imaging Corporation Pa). The results are presentedin FIGS. 5-9.

Example 10 Results of Examples 6-9

[0284] In this series of experiments the zinc finger chimera, was clonedinto the plant expression vector pBA002, placing its expression underthe regulation of CaMVS35 promoter that it is constitutively expressed(see FIG. 3). The zinc finger chimera construct and the reporterconstruct were co-transformed into onion peels by biolistic bombardmentwith DNA-coated gold particles (BioRad, Oxford.UK). Transient expressionof luciferase was recorded after 24 hr expression using an Imagingcamera system. The results are shown in FIG. 5. The results reveal thatexpression of either zinc finger chimera containing VP16 or VP64 wasable to induce strong expression of luciferase.

[0285] When tested with a reporter vector containing one binding site,the zinc finger chimera containing VP16 induced luciferase activity. Inthe absence of the zinc finger chimera the reporter vectors with eithera single copy or tetramer of the minimal binding site alone producedonly background levels of luciferase expression (FIG. 5).

[0286] In a second series of experiments, we used the XVE induciblesystem to express the zinc finger chimera and the reporter construct inonion peels and young Arabidopsis seedlings. The XVE system is anestrogen receptor- based chemical-inducible system for expression ofgenes in transgenic plants (FIG. 3).

[0287] Briefly, a chimeric transcriptional activator XVE is a fusionprotein of the DNA binding domain from the bacterial repressor LexA (X),the acidic transactivator domain VP16 (V) and the regulatory region ofthe human estrogen receptor (E, ER). The zinc finger chimera was clonedinto the multiple cloning site (MCS) of the XVE vector downstream ofeight copies of the LexA operator. The XVE was expressed by constitutivepromoter G10-90 and in the presence of β—17-Estradiol the activated-XVEbinds the LexA operator, inducing the expression of the zinc fingerchimera. Similar experiments were done using the zinc finger chimera XVEconstruct, and the reporter constructs were transformed into two onionpeels, only one of which was sprayed with β—17-Estradiol. The transientexpression of luciferase was recorded after 24 hr expression, but onlythe onion peels sprayed with β—17-estradiol were able to display strongexpression of luciferase (FIG. 6).

[0288] Transformation of onion peels with the reporter vector alonecontaining tetramer of minimal binding site alone did not produce anyluciferase expression with or without of β—17-estradiol. Similar resultswere obtained with a reporter construct containing GFP (FIG. 7). Inaddition, Arabidopsis plants were transformed with pBA4×luciferase andpBA4×luciferase+pER8TFIIIAZifVP16. Only transgenic (T1) lines leavescontaining the zinc finger chimera XVE construct (pER8TFIIIAZifVP16)were able to produce luciferase expression with the addition ofβ—17-Estradiol (FIG. 8).

[0289] In a final experiment of the second series we tested thespecificity of zinc finger chimera by offering two reporter constructscontaining a 27 bp binding sequence upstream of GFP and the other with asingle-mutated one 27 bp sequence binding sequence upstream of the RFP(Red fluorescence protein) cDNA. The single mutation in the 27 bpsequence binding sequence reduced the binding affinity of zinc fingerchimera protein by 10-20 fold. Preliminary experiments done in onionpeels showed a 201 fold difference in the percentage of transformedcells (FIG. 9). These constructs can be co-transformed with theinducible promoter zinc finger chimera into Arabidopsis.

Example 11 The XVE System

[0290] In another series, the Lex A binding domain of the XVE system(pER12) is replaced by the Zinc finger domain from the chimera (see FIG.4) and the 8 LexA operator binding site by 4 minimal binding sites ofthe zinc finger. This new vector ZVE1 is tested by using eitherluciferase or GFP cloned in the same vector in transgenic Arabidopsisplants.

[0291] The use of zinc finger offers the advantages of great specificityand avoids non-specific interaction with the Arabidopsis genome.

[0292] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1 34 1 17 DNA Unknown oligonucleotide sequence used to facilitatetranslation in plants 1 aaggagatat aacaatg 17 2 10 DNA Unknownoligonucleotide sequence used to facilitate translation in plants 2gtcgaccatg 10 3 60 DNA Unknown Oligonucleotide used to prepare aderivative of fd-tet-DOG1 3 ctcctgcagt tggacctgtg ccatggccgg ctgggccgcatagaatggaa caactaaagc 60 4 995 DNA Unknown pTFIIIAZifVP16 4 tctagagcgccgccatggga gagaaggcgc tgccggtggt gtataagcgg tacatctgct 60 ctttcgccgactgcggcgct gcttataaca agaactggaa actgcaggcg catctgtgca 120 aacacacaggagagaaacca tttccatgta aggaagaagg atgtgagaaa ggctttacct 180 cgcttcatcacttaacccgc cactcactca ctcatactgg cgagaaaaac ttcacatgtg 240 actcggatggatgtgacttg agatttacta caaaggcaaa catgaagaag cactttaaca 300 gattccataacatcaagatc tgcgtctatg tgtgccattt tgagaactgt ggcaaagcat 360 tcaagaaacacaatcaatta aaggttcatc agttcagtca cacacagcag ctgccgtatg 420 cttgccctgtcgagtcctgc gatcgccgct tttctcgctc ggatgagctt acccgccata 480 tccgcatccacacaggccag aagcccttcc agtgtcgaat ctgcatgcgt aacttcagtc 540 gtagtgaccaccttaccacc cacatccgca cccacacagg cgagaagcct tttgcctgtg 600 acatttgtgggaggaagttt gccaggagtg atgaacgcaa gaggcatacc aaaatccatt 660 taagacagaaggacgcggcc gcactcgagc ggaattccgg cccaaaaaag aagagaaagg 720 tcgcccccccgaccgatgtc agcctggggg acgagctcca cttagacggc gaggacgtgg 780 cgatggcgcatgccgacgcg ctagacgatt tcgatctgga catgttgggg gacggggatt 840 ccccggggccgggatttacc ccccacgact ccgcccccta cggcgctctg gatacggccg 900 acttcgagtttgagcagatg tttaccgatg cccttggaat tgacgagtac ggtggggaac 960 aaaaacttatttctgaagaa gatctgtaag gatcc 995 5 947 DNA Unknown pTFIIIAZifVP64 5tctagagcgc cgccatggga gagaaggcgc tgccggtggt gtataagcgg tacatctgct 60ctttcgccga ctgcggcgct gcttataaca agaactggaa actgcaggcg catctgtgca 120aacacacagg agagaaacca tttccatgta aggaagaagg atgtgagaaa ggctttacct 180cgcttcatca cttaacccgc cactcactca ctcatactgg cgagaaaaac ttcacatgtg 240actcggatgg atgtgacttg agatttacta caaaggcaaa catgaagaag cactttaaca 300gattccataa catcaagatc tgcgtctatg tgtgccattt tgagaactgt ggcaaagcat 360tcaagaaaca caatcaatta aaggttcatc agttcagtca cacacagcag ctgccgtatg 420cttgccctgt cgagtcctgc gatcgccgct tttctcgctc ggatgagctt acccgccata 480tccgcatcca cacaggccag aagcccttcc agtgtcgaat ctgcatgcgt aacttcagtc 540gtagtgacca ccttaccacc cacatccgca cccacacagg cgagaagcct tttgcctgtg 600acatttgtgg gaggaagttt gccaggagtg atgaacgcaa gaggcatacc aaaatccatt 660taagacagaa ggacgcggcc gcactcgagc ggaattccgg cccaaaaaag aagagaaagg 720tcgaacttca gctgacttcg gatgcattag atgactttga cttagatatg ctaggatctg 780acgcgctaga cgatttcgat ctggacatgt tgggcagcga tgctctagac gatttcgatt 840tagatatgct tggctcggat gccctggatg acttcgacct cgacatgctg tcaagtcagc 900tgagccagga acaaaaactt atttctgaag aagatctgta aggatcc 947 6 14 DNA Unknownsequence incorporating plant translational initiation context sequenceto facilitate translation in plant cells 6 aaggagatat aaca 14 7 29 DNAUnknown target DNA sequence of TFIIIA2ifVP16 and TFIIIAZifVP64 7tgcgtgggcg tgtacctgga tgggagacc 29 8 35 DNA Unknown forward primer usedin PCR to engineer coding region of Zinc finger chimera genes (VP16 andVP64) 8 ccacgcgtcc atgggagaga aggcgctgcc ggtgg 35 9 44 DNA Unknownreverse primer used in PCR to engineer coding region of Zinc fingerchimera genes (VP16 and VP64) 9 ccactagtcc ttacagatct tcttcagaaataagtttttg ttcc 44 10 148 DNA Unknown sense strand primer used inconstruction of reporter plasmids 10 cctctagatc ggtctcccat ccaggtacacgcccacgcaa gtcggtctcc catccaggta 60 cacgcccacg caagtcggtc tcccatccaggtacacgccc acgcaagtcg gtctcccatc 120 caggtacacg cccacgcaag aagcttcc 14811 148 DNA Unknown antisense strand primer used in construction ofreporter plasmids 11 ggaagcttct tgcgtgggcg tgtacctgga tgggagaccgacttgcgtgg gcgtgtacct 60 ggatgggaga ccgacttgcg tgggcgtgta cctggatgggagaccgactt gcgtgggcgt 120 gtacctggat gggagaccga tctagagg 148 12 45 DNAUnknown forward primer used to engineer a single binding site reporterconstruct 12 ccagatctgg tctcccatcc aggtacacgc ccacgcaaga tctcc 45 13 46DNA Unknown reverse primer used to engineer a single binding sitereporter construct 13 ggagatcttg cgtgggcgtg tacctggatg ggagaccaga tctcgg46 14 34 DNA Unknown forward primer used to engineer coding region ofGFP to include NcoI and EcoRI restriction sites 14 ccccatggtg agcaagggcgaggagctgtt cacc 34 15 35 DNA Unknown reverse primer used to engineercoding region of GFP to include NcoI and EcoRI restriction sites 15ccgaattctt acttgtacag ctcgtccatg ccgag 35 16 28 DNA Unknown forwardprimer used to engineer coding region of RFP to include SalI and EcoRIrestriction sites 16 ccctcgagcg gggtaccgcg ggcccggg 28 17 30 DNA Unknownreverse primer used to engineer coding region of RFP to include SalI andEcoRI restriction sites 17 cagttggaat tctagagtcg cggccgctac 30 18 38 DNAUnknown forward primer used to engineer coding region of VP16-estrogenreceptor to include XhoI restriction sites 18 ccgctcgagg cccccccgaccgatgtcagc ctggggga 38 19 38 DNA Unknown reverse primer used to engineercoding region of VP16-estrogen receptor to include XhoI restrictionsites 19 ccgctcgagt attaatttga gaatgaacaa aaaggacc 38 20 38 DNA Unknownforward primer used to engineer coding region of the TFIIIA2if-VP16-estrogen receptor fusion gene to include AseI restriction sites 20gccattaatc ggaatgggag agaaggcgct gccggtgg 38 21 32 DNA Unknown reverseprimer used to engineer coding region of the TFIIIA2if-VP 16-estrogenreceptor fusion gene to include AseI restriction sites 21 gcctattaatttgagaatga acaaaaagga cc 32 22 26 PRT Unknown Consensus zinc fingerstructure 22 Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln Lys SerAsp 1 5 10 15 Leu Val Lys His Gln Arg Thr His Thr Gly 20 25 23 29 PRTUnknown Consensus zinc finger structure 23 Pro Tyr Lys Cys Ser Glu CysGly Lys Ala Phe Ser Gln Lys Ser Asn 1 5 10 15 Leu Thr Arg His Gln ArgIle His Thr Gly Glu Lys Pro 20 25 24 4 PRT Unknown linker sequence usedto join consensus zinc finger motifs 24 Thr Gly Glu Lys 1 25 5 PRTUnknown linker sequence used to join consensus zinc finger motifs 25 ThrGly Glu Lys Pro 1 5 26 5 DNA unknown motif destabilizing messenger RNAs26 attta 5 27 6 DNA unknown motif causing inappropriate polyadenylation27 aataaa 6 28 9 DNA Unknown oligonucleotide sequence used to facilitatetranslation in plants 28 taaacaatg 9 29 9 PRT Unknown sequence bindingto zinc finger phage display library 29 Xaa Xaa Xaa Xaa Xaa Gly Gly CysGly 1 5 30 11 DNA Unknown DNA recognition site for zinc Fingers 1-3 ofTFIIIA 30 ggatgggaga c 11 31 10 DNA Unknown DNA recognition site for thethree zinc fingers of Zif268 31 gcgtgggcgt 10 32 6 DNA Unknown Spacerregion separating zinc finger DNA binding sites 32 gtacct 6 33 36 PRTUnknown amino acid sequence of zinc Finger 4 of TFIIIA includingflanking sequences 33 Asn Ile Lys Ile Cys Val Tyr Val Cys His Phe GluAsn Cys Gly Lys 1 5 10 15 Ala Phe Lys Lys His Asn Gln Leu Lys Val HisGln Phe Ser His Thr 20 25 30 Gln Gln Leu Pro 35 34 107 DNA UnknownNucleotide sequence of Zinc Finger 4 of TFIIIA including flankingsequences 34 aacatcaaga tctgcgtcat gtgtgccatt ttgagaactg tggcaaagcattcaagaaac 60 acaatcaatt aaaggttcat cagttcagtc acacacagca gctgccg 107

1. A method of regulating transcription in a plant cell from a DNAsequence comprising a target DNA operably linked to a coding sequence,which method comprises introducing an engineered zinc finger polypeptideinto said plant cell which polypeptide binds to the target DNA andmodulates transcription of the coding sequence.
 2. The method accordingto claim 1 wherein the target DNA is part of an endogenous genomicsequence.
 3. The method according to claim 1 wherein the target DNA andcoding sequence are heterologous to the cell.
 4. The method according toany one of the preceding claims wherein the zinc finger polypeptide isfused to a biological effector domain.
 5. The method according to claim4 wherein the zinc finger polypeptide is fused to a transcriptionalactivator domain.
 6. The method according to claim 4 wherein the zincfinger polypeptide is fused to a transcriptional repressor domain.
 7. Aplant host cell comprising a polynucleotide encoding an engineered zincfinger polypeptide and a target DNA sequence to which the zinc fingerpolypeptide binds.
 8. A transgenic plant comprising a polynucleotideencoding an engineered zinc finger polypeptide and a target DNA sequenceto which the zinc finger polypeptide binds.
 9. A method according to anyone of claim 1 to 6 wherein the plant cell is part of a plant and thetarget sequence is part of a regulatory sequence to which the nucleotidesequence of interest is operably linked.